U.S. patent application number 11/437734 was filed with the patent office on 2006-09-21 for novel mouse cxc chemokine receptor.
Invention is credited to Hisashi Iizasa, Tadamitsu Kishimoto, Takashi Nagasawa, Toshihiro Nakajima, Kazunobu Tachibana, Nobuaki Yoshida, Osamu Yoshie.
Application Number | 20060211037 11/437734 |
Document ID | / |
Family ID | 14180024 |
Filed Date | 2006-09-21 |
United States Patent
Application |
20060211037 |
Kind Code |
A1 |
Kishimoto; Tadamitsu ; et
al. |
September 21, 2006 |
Novel mouse CXC chemokine receptor
Abstract
The present invention pertains to a DNA encoding a polypeptide
including an entire sequence of the amino acid sequence as shown by
SEQ ID NO: 2 or a partial sequence thereof, or a polypeptide
including the polypeptide described above, wherein any of the
polypeptides has an activity of a receptor capable of binding to a
murine PBSF/SDF-1; a polypeptide encoded by the DNA described
above, wherein the polypeptide has an activity of a receptor
capable of binding to a murine PBSF/SDF-1; cells expressing the
polypeptide described above and a human CD4 protein; and a method
of screening an AIDS onset inhibitor or an HIV-1 infection
inhibitor, characterized by the use of the cells described above.
According to the present invention, there can be provided a novel
murine CXC chemokine receptor gene, a method of screening an HIV-1
infection inhibitor, and the like, each of which is useful in
studies of a therapeutic agent for AIDS and the functional
mechanism of HIV-1 infection.
Inventors: |
Kishimoto; Tadamitsu;
(Osaka, JP) ; Nagasawa; Takashi; (Osaka, JP)
; Tachibana; Kazunobu; (Osaka, JP) ; Iizasa;
Hisashi; (Kanazawa-shi, JP) ; Yoshida; Nobuaki;
(Kashiba-shi, JP) ; Nakajima; Toshihiro; (Osaka,
JP) ; Yoshie; Osamu; (Nishinomiya-shi, JP) |
Correspondence
Address: |
BIRCH STEWART KOLASCH & BIRCH
PO BOX 747
FALLS CHURCH
VA
22040-0747
US
|
Family ID: |
14180024 |
Appl. No.: |
11/437734 |
Filed: |
May 22, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09367052 |
Aug 6, 1999 |
7074616 |
|
|
PCT/JP97/00299 |
Feb 7, 1997 |
|
|
|
11437734 |
May 22, 2006 |
|
|
|
Current U.S.
Class: |
435/6.14 ;
435/5 |
Current CPC
Class: |
C07K 14/7158 20130101;
A61K 38/00 20130101 |
Class at
Publication: |
435/006 ;
435/005 |
International
Class: |
C12Q 1/70 20060101
C12Q001/70; C12Q 1/68 20060101 C12Q001/68 |
Claims
1. A polypeptide having an activity of a receptor that binds to a
murine PBSF/SDF-1, wherein said polypeptide comprises an entire
sequence or partial sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
2. The polypeptide of claim 1, wherein said polypeptide comprises
at least one of deletion, addition, insertion, or substitution of
one or more amino acid residues in SEQ ID NO: 1 or SEQ ID NO:
2.
3. The polypeptide of claim 1, said polypeptide is derived from a
murine pre-B-cell line DW34.
4. A monoclonal antibody against the polypeptide according to claim
1.
5. A pharmaceutical composition for the use as an AIDS onset
inhibitor or an HIV-1 infection inhibitor, comprising a murine
PBSF/SDF-1.
6. A method of screening an AIDS onset inhibitor or an HIV-1
infection inhibitor, said method comprising the steps of: (a)
mixing the cells expressing the polypeptide of claim 1, a human
T-cell-line-tropic HIV-1, and a substance to be screened, and
incubating the resulting mixture; and (b) analyzing localization of
an HIV-1 in the cells.
7. The method according to claim 6, wherein the step of analyzing
localization of an HIV-1 is carried out by using a monoclonal
antibody against a human T-cell-line-tropic HIV-1.
8. A method of screening an AIDS onset inhibitor or an HIV-1
infection inhibitor, said the method comprising the steps of: (a)
mixing the cells expressing the polypeptide of claim 1; cells
expressing an HIV-1 envelope protein; and a substance to be
screened, and incubating the resulting mixture; and (b) determining
a level of the fusion of the above cells with the cells expressing
an HIV-1 envelope protein.
9. A method of screening an AIDS onset inhibitor or an HIV-1
infection inhibitor, or a PBSF/SDF-1 agonist or antagonist, said
method comprising the steps of: (a) mixing the cells expressing the
polypeptide according to claim 1, a murine or human PBSF/SDF-1, and
a substance to be screened, and incubating the resulting mixture;
and (b) determining an intracellular calcium ion level and/or
determining a binding activity of an expressed polypeptide with the
murine or human PBSF/SDF- 1.
10. The method according to claim 9, wherein the antagonist is a
hematopoetic stem cell liberator.
11. A method for detecting an AIDS onset or an HIV-1 infection,
said method comprising the steps of: (a) mixing the cells
expressing the polypeptide of claim 1 with sera, blood cells or
blood of a patient suspected to be infected with an HIV-1, and
incubating the resulting mixture, and (b) analyzing localization of
an HIV-1 in the cells or determining a level of the fusion of the
cells with HIV-1-infected cells.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a 37 C.F.R. .sctn. 1.53(b)
divisional of U.S. application Ser. No. 09/367,052 filed Aug. 6,
1999, which is the National Phase of International Application No.
PCT/JP97/00299 filed Feb. 7, 1997. Each of these applications are
hereby incorporated by reference.
[0002] 1. Technical Field
[0003] The present invention relates to a novel murine CXC
chemokine receptor, and a murine chemokine receptor gene. More
particularly, it relates to a polypeptide encoded by the gene, an
expression vector carrying the gene, a transformant into which the
expression vector is introduced, and a monoclonal antibody against
the polypeptide. Further, it relates to a method for producing the
polypeptide using the transformant. Furthermore, it relates to a
method of screening an agonist or antagonist of chemokines, and a
method of screening an AIDS onset inhibitor or an HIV-1 infection
inhibitor.
[0004] 2. Background Art
[0005] When a tissue impairment takes place owing to causation such
as a bacterial or viral infection, a physical or chemical trauma,
an autoimmune disease, an allergic disease or the like, an
inflammatory reaction accompanied with signs such as flare, edema,
fever and pain is induced, and accumulation and infiltration of
peripheral leukocytes are observed at the local inflammation. The
kinds of the leukocytes infiltrating on the site of an inflammation
vary depending on the diseases. An acute inflammation such as an
ordinary bacterial infection, an immunological complex deposition
and a trauma involves accumulation and infiltration mainly of a
neutrophile; a tubercular infection, a typhoid infection and a
delayed hypersensitivity involve those mainly of a monocyte; and a
viral infection involves those mainly of a lymphocyte, while an
eosinophile and a basophile infiltrate accompanied with an
immediate allergy and a parasite infection [Baggioloni, M. et al.,
Immunol. Today, 15, 127-133 (1994)]. Recently, there have been
found that chemotactic factors of polypeptides having certain
degrees of selectivity to leukocytes having chemotactic activities,
the polypeptides having characteristic four cysteine residues.
Since they are in a family, members of which are homologous in
their amino acid sequences and related to each other also in terms
of the biological activities, they are referred to as chemokines
(having chemoattractant and cytokine activity) [Lindley, I. J. D.
et al., Immunol. Today, 14-24 (1993)].
[0006] Four cysteine residues of a chemokine are linked with
disulfide bonds between the first and third residues and between
the second and fourth residues, respectively. Since their
characteristics are found on the biological activities in which
whether or not one additional amino acid is contained between the
first and second cysteine residues, their subfamilies are
distinguished by referring to as CXC chemokines and CC chemokines
[Baggioloni, M. et al., Adv. Immunol., 55, 97-179 (1994)].
[0007] The CXC chemokines which have been found so far are
PBSF/SDF-1; IL-8 [Yoshimura, T. et al., Proc. Natl. Acad. Sci.
U.S.A., 84, 9233-9237 (1987)]; NAP-2 [Walz, A. et al., Biochem.
Biophys. Res. Commun., 159, 969-975 (1989)]; NAP-4; GRO.alpha.
[Richmondo, A. et al., J Cell. Biochem., 36, 185-198 (1988)];
GRO.beta. [Haskill, S. et al., Proc. Natl. Acad. Sci. U.S.A., 87,
77732-7736 (1990)]; GRO.gamma. [Haskill, S. et al., (1990) ibid];
GCP-2 [Proos t, P. et al., J Immunol., 150, 1000-1010 (1993)];
ENA-78 [Wayz, A. et al., J Exp. Med., 174, 1355-1362 (1991)]; PF-4
[Deuel, T. F. et al., Proc. Natl. Acad. Sci. U.S.A., 74, 2256-2258
(1977)]; a human CXCR4/fusin/HUMSTSR [Feng, Y. et al., Science,
272, 872-877 (1996)]; and IP-10 [Dewald, B. et al., Immunol. Lett.,
32, 81-84 (1992)].
[0008] And the CC chemokines are MCP-1 [Yoshimura, T. et al., J
Immunol., 142, 1956-1962 (1989)]; MCP-2 [Chang, H. C. et al., Int.
Immunol., 1, 388-397 (1989)]; MCP-3 [Van Damme, J. et al., J Exp.
Med, 176, 59-65 (1992)]; MIP-1.alpha. [Obaku, K. et al., J
Biochem., 99, 885-894 (1986)]; MIP-1.beta. [Lipes, M. A. et al.,
Proc. Natl. Acad. Sci. US.A., 85, 9704-9708 (1988)]; RANTES
[Schall, T. et al., J Immunol., 141, 1018-1025 (1988)]; I-309
[Miller, M. D. et al., J Immunol., 143, 2907-2916 (1989)]; and
eotaxin [Jose, P. et al., J Exp. Med, 179, 881-887 (1994)].
[0009] Most of the CXC chemokines have chemotactic activities on a
neutrophile but not on a monocyte. Most of the CC chemokines have
chemotactic activities on a monocyte but not on a neutrophile. In
addition, as to other leukocytes such as an eosinophile, a
basophile and a lymphocyte, there have been reported to have the
chemotactic activities for some of CXC and CC chemokines. While CC
chemokines including RANTES, MIP-1.alpha. and MCP-1, and IL-8,
which is a CXC chemokine, have been found to possess chemotactic
activities on human lymphocytes, none of them are chemotactic
factors specific to lymphocytes.
[0010] It has been reported that murine PBSF/SDF-1 is a CXC
chemokine which is identified as a murine pre-B-cell
growth-stimulating factor originally secreted from a murine bone
marrow stromal cell line PA6 with its amino acid sequence (FIG. 1)
(SEQ ID NO:22) [Nagasawa, T. et al., Proc. Natl. Acad. Sci. USA,
91, 2305-2309 (1994)]. In addition, recently, it has been clarified
to have a potent chemotactic activity also on a human T lymphocyte
[Bleul, C. et al., J Exp. Med., 184, 1101-1110].
[0011] Various studies have been conducted on receptors for
chemokines. There have been reported IL-8RA, which is a receptor
specific to IL-8; IL-8RB, which is a receptor for IL-8 and other
CXC chemokines; CC CKR1, which is a receptor specific to
MIP-1.alpha. and RANTES; CC CKR2A, which is a receptor specific to
MCP-1; CC CKR2B, which is a receptor specific to MCP-1 and MCP-3;
CC CKR3, which is a receptor specific to eotaxin, MCP-3, and RANTES
[Combadiere, C. et al., J Biol., 270, 16491-16494 (1995)]; and CC
CKR5, which is a receptor specific to MIP-1.alpha., MIP-1.beta. and
RANTES. Recently, CXCR4/fusin/HUMSTSR has been identified as a
receptor for SDF-1 which is a human CXC chemokine.
[0012] In addition, among the chemokine receptors mentioned above,
there has been clarified that CC CKR5, CC CKR2B, CC CKR3 and
CXCR4/fusin/HUMSTSR have functions as receptors for HIV-1 by acting
cooperatively with CD4, a protein present on a cell membrane, and
that an infection with HIV-1 mediated by each receptor is inhibited
by the ligands of these receptors.
[0013] Two characteristically different HIV-1s are involved in the
infection with an HIV-1, which is an AIDS-causing virus, and in the
onset of AIDS. A monocyte-tropic HIV-1 with which monocytes,
macrophages and T lymphocytes are mainly infected is involved in
the viral proliferation in a human body during the period of
infection and latent infection, and a T-cell-line-tropic HIV-1 with
which T lymphocytes are mainly infected is involved in the
reduction of the number of T lymphocytes and the onset of AIDS. In
order to infect cells with the two HIV-1s mentioned above, two
receptors are required. One is CD4 protein, which is a cell
membrane protein, and is a common receptor for the two HIV-1s
mentioned above. The other is a protein referred to as a coreceptor
which has an activity as a receptor by acting cooperatively with
the CD4 protein, and is specific to each of the two HIV-1s.
[0014] Recently, there has been clarified that a coreceptor for a
main monocyte-tropic HIV-1 is found to be CC CKR5, which is a CC
chemokine receptor, and a coreceptor for T-cell-line-tropic HIV-1
is found to be human CXCR4/fusin/HUMSTSR, which is a CXC chemokine
receptor. Further, there has been clarified that the infection with
a monocyte-tropic HIV-1 is inhibited by MIP-1.alpha., MIP-1.beta.
and RANTES, which are CC CKR5 ligands, and the infection with a
T-cell-tropic HIV-1 is inhibited by a human PBSF/SDF-1, which is a
human CXCR4/fusin/ UMSTSR ligand, suggesting that the chemokine
receptors described above could be a target of an HIV-1 infection
inhibitor.
[0015] On the other hand, which domain in a human
CXCR4/fusin/HUMSTSR is essential for the infection with a
T-cell-line-tropic HIV-1 has not been identified so far. A CXC
chemokine receptor, human CXCR4/fusin/HUMSTSR is a seven
transmembrane-spanning-domain receptor, and a three-dimensional
structure formed by four extracellular domains is considered to be
significant in the binding with a ligand or an HIV-1. For the
purpose of identifying a functional domain of a human
CXCR4/fusin/HUMSTSR, it is necessary to produce a
CXCR4/fusin/HUMSTSR variant so as to maintain the three-dimensional
structure as a receptor. The identification of a functional domain
of a human CXCR4/fusin/HUMSTSR is extremely useful in the
development of an HIV-1 infection inhibitor.
[0016] In addition, the elucidation of the mechanism for causation
of an HIV-1 species-specificity is significant in the development
of an HIV-1 infection model animal, as in the case of clarifying an
intracellular factor required for a viral infection. Although a
mouse is an excellent experimental animal which can easily be
handled and obtained at a low cost of which characteristics are
clarified in detail, there has been no report that a mouse is
infected with an HIV-1.
[0017] Murine cells have several barriers concerning an HIV-1 viral
infection. A first barrier is present on the stage where a virus is
bound to murine cells. A human CD4 is bound to an HIV-1, but a
murine CD4 is not bound to an HIV-1. However, there has been
clarified in the existing studies that when a human CD4 is
expressed in vitro on the surface of murine cell lines including a
T-cell line, the adsorption of an HIV to the cells takes place, but
an entry into the cells does not. It was clarified from the above
results that the murine cells expressing a human CD4 do not support
an entry of the virus, suggesting that there is a receptor, in
addition to CD4, which is human-specific and essential for a
membrane fusion (which takes place at an entry of the virus) of
which molecule is absent in murine cells.
[0018] The HIV-1 is recognized to have a difference in an infection
ability to CD 4-positive cells depending on the strains. Some
strains are classified as monocyte- or macrophage-tropic (M-tropic)
strains since they infect a monocyte, and others are classified as
T-cell-line-tropic (T-tropic) strains since they infect a T-cell
line.
[0019] As an HIV-1 infection is progressed, a monocyte-tropic virus
observed frequently at an initial stage of the infection is
replaced with a T-cell-line-tropic virus. In 1996, there has been
reported that CXCR4/fusin, which is a seven
transmembrane-spanning-domain G protein-coupled receptor, is
essential for the entry of a T-cell-line-tropic HIV-1 into human
CD4-positive cells. These results urged the present inventors to
study whether or not the function of the CXCR4 as a viral entry
receptor is species-specific.
DISCLOSURE OF THE INVENTION
[0020] The present inventors have clarified that a murine CXCR4 as
a receptor of a murine PBSF/SDF-1, which is one of CXC chemokines,
is isolated, and found its amino acid sequence to be in 90%
identity with a human CXCR4. The present inventors have established
cells transfected with a human CD4 and a murine CXCR4, and studied
whether or not a human CXCR4, which is an HIV-1 receptor, serves as
a barrier against an HIV-1 entry present in the murine cells.
[0021] Accordingly, an object of the present invention is to
provide a novel murine CXC chemokine receptor gene; a polypeptide
encoded by the gene; an expression vector carrying the gene; a
transformant harboring the expression vector; a monoclonal antibody
against the polypeptide; a method for producing the polypeptide;
and a method of screening an agonist or antagonist of the
polypeptide and also a method of screening an HIV-1 infection
inhibitor, each of which is useful in studies of a therapeutic
agent for AIDS and the functional mechanism of HIV-1 infection.
[0022] Specifically, as a result of intensive studies in order to
solve the problems described above, the present inventors have
succeeded in cloning a novel murine chemokine receptor gene from a
murine pre-B-cell line DW34 of which growth is enhanced depending
on a murine PBSF/SDF-1. The present inventors have also found that
cells expressing murine CXCR4 and human CD4 fuse with cells
expressing an env protein derived from a T-cell-line-tropic HIV-1
strain, and those cells are infected with such a T-cell-line-tropic
HIV-1 strain. These results lead to a conclusion that CXCR4 is not
a species-specific barrier against the entry of a
T-cell-line-tropic HIV-1, which is present in the murine cells. In
addition, because of the fact that a T-cell-line-tropic HIV-1
chimera virus clone of which env or V3 region is replaced with that
of a monocyte-tropic HIV-1 does not infect cells expressing murine
CXCR4 and human CD4 cells, there has been revealed that a V3 loop
of an HIV-1 envelope protein is essential for a murine
CXCR4-mediated HIV-1 entry. Based on these findings, the present
invention has been completed.
[0023] In sum, the present invention pertains to: [0024] (1) a DNA
encoding a polypeptide comprising an entire sequence of the amino
acid sequence as shown by SEQ ID NO: 2 or a partial sequence
thereof, or a polypeptide comprising the polypeptide described
above, wherein any of the polypeptides has an activity of a
receptor capable of binding to a murine PBSF/SDF-1; [0025] (2) a
DNA encoding a polypeptide resulting from at least one of deletion,
addition, insertion, or substitution of one or more amino acid
residues in an entire sequence of the amino acid sequence as shown
by SEQ ID NO: 2 or a partial sequence thereof, wherein any of the
polypeptides has an activity of a receptor capable of binding to a
murine PBSF/SDF-1; [0026] (3) a DNA comprising an entire sequence
of the nucleotide sequence as shown by SEQ ID NO: 1 or a partial
sequence thereof, or a DNA comprising the DNA described above,
wherein any of the DNAs encodes a polypeptide having an activity of
a receptor capable of binding to a murine PBSF/SDF-1; [0027] (4) a
DNA resulting from at least one of deletion, addition, insertion,
or substitution of one or more bases in a DNA comprising an entire
sequence of the nucleotide sequence as shown by SEQ ID NO: 1 or a
partial sequence thereof, or a DNA comprising the DNA, wherein any
of the DNAs encodes a polypeptide having an activity of a receptor
capable of binding to a murine PBSF/SDF-1; [0028] (5) a DNA being
capable of hybridizing under stringent conditions with the DNA of
any one of items (1) to (4) above, and encoding a polypeptide
having an activity of a receptor capable of binding to a murine
PBSF/SDF-1; [0029] (6) a polypeptide encoded by the DNA of any one
of items (1) to (5) above, wherein the polypeptide has an activity
of a receptor capable of binding to a murine PBSF/SDF-1; [0030] (7)
a polypeptide comprising an entire amino acid sequence as shown by
SEQ ID NO: 2 or a partial sequence thereof, or a polypeptide
comprising the polypeptide described above, wherein any of the
polypeptides has an activity of a receptor capable of binding to a
murine PBSF/SDF-1; [0031] (8) a polypeptide resulting from at least
one of deletion, addition, insertion, or substitution of one or
more amino acid residues in an entire amino acid sequence as shown
by SEQ ID NO: 2 or a partial sequence thereof, wherein the
polypeptide has an activity of a receptor capable of binding to a
murine PBSF/SDF- 1; [0032] (9) the polypeptide according to any one
of items (6) to (8) above, derived from a murine pre-B-cell line
DW34; [0033] (10) an expression vector carrying the DNA according
to any one of items (1) to (5) above; [0034] (11) a transformant
obtained by introducing the expression vector according to item
(10) above into a host; [0035] (12) the transformant according to
item (11) above, wherein the host is a mammalian cell line; [0036]
(13) a method for producing a polypeptide having an activity of a
receptor capable of binding to a murine PBSF/SDF-1, characterized
in that the method comprises culturing the transformant according
to item (11) or (12) above under conditions capable of expressing
the expression vector according to item (10) above; [0037] (14) a
monoclonal antibody against the polypeptide according to any one of
items (6) to (9) above; [0038] (15) a pharmaceutical composition
for the use as an AIDS onset inhibitor or an HIV-1 infection
inhibitor, comprising a murine PBSF/SDF-1; [0039] (16) cells
expressing the polypeptide according to any one of items (6) to (9)
above and a human CD4 protein; [0040] (17) a method of screening an
AIDS onset inhibitor or an HIV-1 infection inhibitor, characterized
in that the method comprises the steps of: [0041] (a) mixing the
cells expressing the polypeptide according to any one of items (6)
to (9) above, or cells according to item (16) above; a human
T-cell-line-tropic HIV-1; and a substance to be screened, and
incubating the resulting mixture; and [0042] (b) analyzing
localization of an HIV-1 in the cells; [0043] (18) the method
according to item (17) above, wherein the step of analyzing
localization of an HIV-1 is carried out by using a monoclonal
antibody against a human T-cell-line-tropic HIV-1; [0044] (19) a
method of screening an AIDS onset inhibitor or an HIV-1 infection
inhibitor, characterized in that the method comprises the steps of:
[0045] (a) mixing the cells expressing the polypeptide according to
any one of items (6) to (9) above, or cells according to item [16]
above; cells expressing an HIV-1 envelope protein; and a substance
to be screened, and incubating the resulting mixture; and [0046]
(b) determining a level of the fusion of the above cells with the
cells expressing an HIV-1 envelope protein; [0047] (20) a method of
screening an AIDS onset inhibitor or an HIV-1 infection inhibitor,
or a PBSF/SDF-1 agonist or antagonist, characterized in that the
method comprises the steps of: [0048] (a) mixing the cells
expressing the polypeptide according to any one of items (6) to (9)
above, or cells according to item (16) above; a murine or human
PBSF/SDF-1; and a substance to be screened, and incubating the
resulting mixture; and [0049] (b) determining an intracellular
calcium ion level and/or determining a binding activity of an
expressed polypeptide with the murine or human PBSF/SDF-1; [0050]
(21) the method according to item (20) above, wherein the
antagonist is a hematopoetic stem cell liberator; [0051] (22) a kit
for detecting an AIDS onset or an HIV-1 infection, comprising the
cells expressing the polypeptide according to any one of items
items (6) to (9) above, or cells according to item (16) above; and
[0052] (23) a method for detecting an AIDS onset or an HIV-1
infection, characterized in that the method comprises; [0053] (a)
mixing the cells expressing the polypeptide according to any one of
items items (6) to (9) above, or cells according to item (16) above
with sera, blood cells or blood of a patient suspected to be
infected with an HIV-1, and incubating the resulting mixture, and
[0054] (b)analyzing localization of an HIV-1 in the cells or
determining a level of the fusion of the cells with HIV-1-infected
cells.
BRIEF DESCRIPTION OF THE DRAWINGS
[0055] FIG. 1 shows a nucleotide sequence of a murine PBSF/SDF-1
cDNA (SEQ ID NO: 21), and an amino acid sequence (SEQ ID NO: 22) of
a murine PBSF/SDF-1 encoded by the above nucleotide sequence.
[0056] FIG. 2 shows electrophoretic results of Example 2 by means
of Northern blotting method, wherein A shows the results on mRNA of
murine tissues; and B shows the results on mRNA of murine
fetus.
[0057] FIG. 3 is graphs each showing results of Example 6, wherein
the abscissa indicates the passage of period of time, and the
ordinate indicates the ratio of fluorescence intensities
([fluorescence intensity at 340 nm]/[fluorescence intensity at 380
nm]). The cells used are CHO cells in which the chemokine receptor
is not expressed.
[0058] FIG. 4 is graphs each showing results of Example 6, wherein
the abscissa indicates the passage of period of time, and the
ordinate indicates the ratio of fluorescence intensities
([fluorescence intensity at 340 nm]/[fluorescence intensity at 380
nm]). The cells used are CHO cells in which the human chemokine
receptor CC CKR2B is expressed.
[0059] FIG. 5 is graphs each showing results of Example 6, wherein
the abscissa indicates the passage of period of time, and the
ordinate indicates the ratio of fluorescence intensities
([fluorescence intensity at 340 nm]/[fluorescence intensity at 380
nm]). The cells used are CHO cells in which the receptor (murine
CXCR4) of murine chemokine (PBSF/SDF-1) is expressed.
[0060] FIG. 6 is graphs each showing results of Example 6, wherein
the abscissa indicates the passage of period of time, and the
ordinate indicates the ratio of fluorescence intensities
([fluorescence intensity at 340 nm]/[fluorescence intensity at 380
nm]). The cells used are CHO cells in which the human chemokine
receptor CXCR4/fusin/HUMSTSR is expressed.
[0061] FIG. 7 is graphs each showing results of Example 6, wherein
the abscissa indicates the passage of period of time, and the
ordinate indicates the ratio of fluorescence intensities
([fluorescence intensity at 340 nm]/[fluorescence intensity at 380
nm]). The cells used are CHO cells in which a murine CXCR4 is
expressed.
[0062] FIG. 8 is a graph showing that a murine CXCR4 supports a
membrane fusion via env protein derived from a human
T-cell-line-tropic HIV-1 strain. NIH3T3, which is target cells, is
subjected to infection with a recombinant vaccinia virus in which
human CD4, T7 polymerase, and an .omega.-subunit of .beta.-gal are
expressed. After infection, these cells are transfected with a
murine CXCR4, a human CXCR4, or a human CCR5. HeLaS3, which is
effector cells, is subjected to infection with a recombinant
vaccinia virus in which env protein derived from NL 432 or SF 162
and an .alpha.-subunit of .beta.-gal are expressed. After the
resulting infected cells are subjected to cell fusion, the
resulting fusion product is subjected to .beta.-gal assay.
[0063] FIG. 9 is a graph showing that a murine CXCR4 supports an
infection with a human T-cell-line-tropic HIV-1 virus. SW480 cells
(A) are transfected with a human CD4, and each of chemokine
receptors (murine CXCR4, human CXCR4, human CCR5, and human CCR2b).
The resulting transfected cells are subjected to infection with
each of NL432 strain, IIIB strain and SF162 strain of HIV-1. A cell
lysate of each of the resulting infected cells is then subjected to
.beta.-gal assay.
[0064] FIG. 10 is a graph showing that a murine CXCR4 supports an
infection with a human T-cell-line-tropic HIV-1 virus. HOS cells
(B) are transfected with a human CD4, and each of chemokine
receptors (murine CXCR4, human CXCR4, human CCR5, and human CCR2b).
The resulting transfected cells are subjected to infection with
each of NL432 strain, IIIB strain and SF162 strain of HIV-1. A cell
lysate of each of the resulting infected cells is then subjected to
.beta.-gal assay.
[0065] FIG. 11 is a graph showing that a murine CXCR4 supports an
infection with a human T-cell-line-tropic HIV-1 virus. U87MG cells
(C) are transfected with a human CD4, and each of chemokine
receptors (murine CXCR4, human CXCR4, human CCR5, and human CCR2b).
The resulting transfected cells are subjected to infection with
each of NL432 strain, IIIB strain and SF162 strain of HIV-1. A cell
lysate of each of the resulting infected cells is then subjected to
.beta.-gal assay.
[0066] FIG. 12 is a schematic view showing the structure of
chimeric provirus clone, wherein env or V3 loop of SF162 is
incorporated into a provirus DNA of NL432, which is a human
T-cell-line-tropic HIV-1 strain, wherein E denotes EcoRI; Ba
denotes BamHI; St denotes Stul; and Nh denotes NheI.
[0067] FIG. 13 is a diagram showing that a V3 loop of an envelope
protein gp 120 is essential in an entry of HIV-1 via a murine
CXCR4. The SW480 cells expressing the human CD4 and the receptors
shown in the figure are subjected to infection with NL432 strain
and SF162 strain of HIV-1, and NL432 env-162 and NL432 V3-162 ,
which are chimeric provirus clones.
BEST MODE FOR CARRYING OUT THE INVENTION
DNA of the Present Invention
[0068] The DNA of the present invention is not particularly limited
as long as it is a DNA encoding a murine PBSF/SDF-1 receptor
(murine CXCR4), which is a novel murine CXC chemokine receptor.
Concretely, the following DNAs are exemplified: [0069] 1) a DNA
encoding a polypeptide comprising an entire sequence of the amino
acid sequence as shown by SEQ ID NO: 2 or a partial sequence
thereof, or a polypeptide comprising the polypeptide described
above, wherein any of the polypeptides has an activity of a
receptor capable of binding to a murine PBSF/SDF-1
[0070] 2) a DNA encoding a polypeptide resulting from at least one
of deletion, addition, insertion, or substitution of one or more
amino acid residues in an entire sequence of the amino acid
sequence as shown by SEQ ID NO: 2 or a partial sequence thereof,
wherein any of the polypeptides has an activity of a receptor
capable of binding to a murine PBSF/SDF- 1; [0071] 3) a DNA
comprising an entire sequence of the nucleotide sequence as shown
by SEQ ID NO: 1 or a partial sequence thereof, or a DNA comprising
the DNA, wherein any of the DNAs encodes a polypeptide having an
activity of a receptor capable of binding to a murine PBSF/SDF-1;
[0072] 4) a DNA resulting from at least one of deletion, addition,
insertion, or substitution of one or more bases in a DNA comprising
an entire sequence of the nucleotide sequence as shown by SEQ ID
NO: 1 or a partial sequence thereof, or a DNA comprising the DNA,
wherein any of the DNA encodes a polypeptide having an activity of
a receptor capable of binding to a murine PBSF/SDF-1; and [0073] 5)
a DNA being capable of hybridizing under stringent conditions with
the DNA of any one of items 1) to 4) above, and encoding a
polypeptide having an activity of a receptor capable of binding to
a murine PBSF/SDF-1.
[0074] In addition, in item 2), the phrase "deletion, addition,
insertion, or substitution of one or more amino acid residues" is
not particularly limited, which, for instance, refers to deletion,
addition, insertion, or substitution of one or several amino acid
residues. Here, the term "several" refers, for instance, to a
number of 10 or less. Further, in item 4), the extent of deletion,
addition, insertion, or substitution of the bases of the DNA of the
present invention is one or more bases, preferably one to several
bases. Here, the term "several" refers, for instance, to a number
of 10 or less. In addition, as long as the function or activity of
the polypeptide to be expressed is of the same level, there may be
included amino acid residues or bases which are chemically or
biochemically modified, or non-naturally occurring or
derivatized.
[0075] The DNA of the present invention can be isolated by
amplifying a nucleotide sequence having homology with a known
chemokine receptor by PCR, and screening a murine cDNA library
using the amplified fragment as a probe.
[0076] An experimental method which can be employed in the present
invention involves general procedures employed in molecular biology
(DNA electrophoresis, a method of recovery of an
electrophoretically separated DNA from a gel, ligation, host
transformation, culture of recombinant host, plasmid DNA
preparation, DNA cleavage with restriction enzymes, DNA
radiolabelling and the like) which are well known to one of
ordinary skill in the art, including, for instance, those described
in Molecular Cloning 2nd Ed. [Maniatis et al., Cold Spring Harbor
Laboratory, New York (1989)].
[0077] The primer used in PCR includes those obtained on the basis
of an amino acid sequence conserved in a reported human chemokine
receptor, and, for example, added with an appropriate restriction
enzyme site on a 5'-side of a condensed forward primer to a DNA
sequence encoding an amino acid sequence of a second
transmembrane-spanning domain; or added with an appropriate
restriction enzyme site on a 5'-side of a condensed reverse primer
to a DNA sequence encoding an amino acid sequence of a seventh
transmembrane-spanning domain. These primers can be synthesized
with a DNA synthesizer.
[0078] Also, the murine mRNA used in a cDNA cloning can be purified
from cells from, for instance, a murine pre-B-cell line DW34
(provided by Prof. Nishikawa of Kyoto Univ.), and the like with a
commercially available mRNA purification kit.
[0079] In addition, the murine genomic DNA cloning can be performed
by using, for instance, a DNA fragment derived from a cDNA of a
murine CXCR4, and the like.
[0080] The nucleotide sequence of a cDNA or the nucleotide sequence
of a genomic DNA thus obtained is subjected to its nucleic acid
homology search, referring to, for example, GenBank/EMBL/DDBJ DNA
sequence data base, and whereby whether or not the cDNA obtained
encodes a chemokine receptor can be deduced. SEQ ID NO: 1 in
Sequence Listing shows the nucleotide sequence of the cDNA
obtained. Since the nucleotide sequence spanning from 120-position
to 1196-position in SEQ ID NO: I is the longest open reading frame,
the amino acid sequence (SEQ ID NO: 2) deduced on the basis of the
nucleotide sequence of this open reading frame is subjected to its
homology search, using a program such as DNASIS (HITACHI, LTD.) or
BLAST [Altschul, F. et al., J Mol. Biol., 215, 403-410], to a
database such as Genbank, EMBL or DDBJ, whereby the polypeptide
encoded by the DNA of the present invention can be further
studied.
[0081] As a result, the polypeptide having the amino acid sequence
as shown by SEQ ID NO: 2 has been deduced to be a trimer G
protein-coupled receptor covering a seven transmembrane-spanning
domain, characteristic to a chemokine receptor. In addition, as a
result of comparison with the amino acid sequences of known CXC
chemokine receptors, there has been revealed that a human
CXCR4/fusin/HUMSTSR is most closely resembles it (90%
identity).
[0082] In addition, since cells in which the DNA of the present
invention is expressed have had receptor activity to a chemokine
(murine PBSF/SDF-1) as well as an intracellular calcium
level-increasing activity, the DNA of the present invention has
been found to encode the novel murine chemokine receptor, and the
protein encoded by this DNA is named a murine CXCR4.
[0083] The term "chemokine" refers to, among causative substances
for which leukocytes show chemotactic activity to the local
inflammation reaction as described above, a family of polypeptides
having certain degrees of selectivity for migrating leukocytes and
having four characteristic cysteine residues. These polypeptides
are related with each other in their amino acid sequences and the
biological activities. Four cysteine residues of a chemokine form
disulfide bonds respectively between the first and third residues
and between the second and fourth residues. The chemokine carrying
another amino acid between the first and second cysteine residues
is referred to as a "CXC chemokine," which is differentiated from
the chemokine which has no additional amino acids referred to as
"CC chemokine." Generally, there has been known that the CC
chemokine has a chemotactic activity on a monocyte, but not on a
neutrophile, and that the CXC chemokine has a chemotactic activity
on a neutrophile, but not on a monocyte.
[0084] The term "chemokine receptor" refers to a family of cell
membrane proteins bound specifically to the chemokines described
above. The chemokine receptors are related with each other in their
amino acid sequences and structures. All of the chemokine receptors
have a seven transmembrane-spanning-domain characteristic to a
rhodopsin family and a binding domain with a trimer G protein. The
chemokine receptors are classified into two subgroups on the basis
of the specificities to ligands. Among the chemokines described
above, one bound specifically to the CXC chemokine is referred to
as a "CXC chemokine receptor," which is differentiated from a "CC
chemokine receptor" which is bound specifically to the CC
chemokine. Generally, a chemokine receptor has an intracellular
calcium level-increasing activity when bound to the respective
ligand. Recently, there have been clarified that some chemokine
receptors have not only an activity as a chemokine receptor but
also an activity as an HIV-1 receptor by acting cooperatively with
a molecule called CD4 which is present on a cell membrane.
[0085] In the present specification, the receptor activity to a
murine PBSF/SDF-1 can be determined, for instance, in a manner as
described below.
[0086] A PBSF/SDF-1 peptide which is a murine CXCR4 ligand is
labeled with .sup.125I using, for example, BOLTON-HUNTER reagent,
or is labeled with an enzyme such as an alkaline phosphatase. The
labeled PBSF/SDF-1 peptide is added to a suspension of cells
expressing a polypeptide having receptor activity, and incubated at
a given temperature. After washing, the amount of the PBSF/SDF-1
peptide bound to the cells can be determined by quantifying the
label, whereby assaying the receptor activity. Examples of the
cells used herein are a murine pre-B-cell line DW34; human fetal
kidney cell line 293 cells, or CHO cells derived from a Chinese
hamster ovary cell line treated so as to express a murine CXCR4,
and the like.
[0087] In addition, it is preferable that the polypeptide of the
present invention has an activity of increasing the level of the
intracellular calcium ions when bound to the ligand. The above
activity can, for instance, be determined as described below.
[0088] The cells expressing the polypeptide, the subject for
measurement of the above activity, are washed with a buffer, and
the washed cells are suspended in an appropriate buffer
[comprising, for instance, HBSS (20 mM Hepes, 125 mM NaCl, 5 mM
KCl, 1 mM MgCl.sub.2, 0.5 mM glucose, and 0.1% BSA at pH 7.4)]. A
fluorescence reagent which is likely to be affected by the
intracellular calcium ions is added to the suspension and
incubated, so that the cells can be labeled. The labeled cells are
washed with a buffer, and subsequently suspended in an appropriate
buffer, so that the activity can be determined from the changes in
the fluorescence intensities when a chemokine, which is a ligand,
is added.
[0089] For instance, when fura-PE3AM (Texas Fluorescence
Laboratories) is used as a fluorescence reagent, the determination
is taken under conditions such that excitation wavelengths are 340
nm and 380 nm, a fluorescence wavelength is 510 nm, and a response
is 0.5 seconds. Thereafter, the ratio of [fluorescence intensity at
an excitation wavelength of 340 nm] to [fluorescence intensity at
an excitation wavelength of 380 nm] is calculated. When the level
of the intracellular calcium ions is increased in the cells to be
measured by the addition of a chemokine, an increase in the ratio
of the fluorescence intensities can be found. In addition, by
adding different kinds of chemokines, the receptor specificities to
the ligands can be also confirmed.
[0090] In addition, the presence of the mRNA in the murine CXCR4
can be confirmed by employing a usual mRNA specific detection
method. For instance, the mRNA can be detected by Northern blotting
analysis or in situ hybridization method by using an antisense RNA
or cDNA as a probe. Alternatively, the mRNA can be also detected by
converting an mRNA to a cDNA with a reverse transcriptase, and then
performing PCR by an appropriate combination of primers.
Polypeptide of the Present Invention
[0091] The polypeptide of the present invention includes, for
instance, the following: [0092] 1) a polypeptide encoded by the DNA
of the present invention, wherein the polypeptide has an activity
of a receptor capable of binding to a murine PBSF/SDF; [0093] 2) a
polypeptide comprising an entire amino acid sequence as shown by
SEQ ID NO: 2 or a partial sequence thereof, or a polypeptide
comprising the polypeptide described above, wherein any of the
polypeptides has an activity of a receptor capable of binding to a
murine PBSF/SDF; [0094] 3) a polypeptide resulting from at least
one of deletion, addition, insertion, or substitution of one or
more amino acid residues in an entire amino acid sequence as shown
by SEQ ID NO: 2 or a partial sequence thereof, wherein the
polypeptide has an activity of a receptor capable of binding to a
murine PBSF/SDF; and [0095] 4) the polypeptide according to any one
of items 1) to 3) above, derived from a murine pre-B-cell line
DW34.
[0096] In Embodiment 3), the extent of deletion, addition,
insertion, or substitution of the amino acid residues of the
polypeptide of the present invention is one or more, and the number
of mutation is not particularly limited as long as the polypeptide
has an activity of a receptor capable of binding to a murine
PBSF/SDF-1. For instance, the number of mutations may be from one
to several. Here, the term "several" refers to a number of, for
instance, 10 or less. In addition, as long as the function or
activity of the polypeptide is of the same level, there may be
encompassed amino acid residues which are chemically or
biochemically modified, or non-naturally occurring or
derivatized.
[0097] In addition, it is preferable that the polypeptide of the
present invention is those derived from a murine pre-B-cell line
DW34.
[0098] The presence of the polypeptide of the present invention can
be confirmed by employing a usual detection method for a specific
protein, including, for instance, usual immunoprecipitation method,
Western blotting method, or analysis by FACS each using an antibody
specific to a murine CXCR4.
Expression Vector and Transformant of the Present Invention
[0099] The expression vector of the present invention can be
obtained by, for example, incorporating the DNA of the present
invention into a known vector such as pEFBOS, pCAGGStkNeo, or
pMX.
[0100] In addition, the transformant of the present invention can
be obtained by introducing the expression vector of the present
invention into a desired host. The host is not particularly
limited, and is preferably a mammalian cell line. Examples of the
mammalian cell line are a murine pre-B-cell line, a human fetal
kidney cell line, a cell line derived from a Chinese hamster ovary,
and the like, and the cell line derived from a hamster ovary is
preferable. A method for introducing an expression vector into a
host may be any known method, including, for instance, such as a
calcium phosphate method, a DEAE dextran method and an
electroporation method.
[0101] In addition, the transformant described above is cultured
under conditions capable of expressing the expression vector,
whereby producing a polypeptide having an activity of a receptor
capable of binding to the murine PBSF/SDF-1. The polypeptide
produced in the manner described above can readily be purified by a
usual column chromatography or an affinity chromatography using the
antibody of the present invention.
Monoclonal Antihody of the Present Invention
[0102] Examples of the monoclonal antibody of the present invention
are ones against the murine CXCR4 polypeptide and ones against a
fusion protein of the above polypeptide with a human
CXCR4/fusin/HUMSTSR.
[0103] The above monoclonal antibody can be prepared by a method
described below.
[0104] As an immunogen, there are employed a synthetic polypeptide
prepared by a usual peptide synthesizer based on a part of the
amino acid sequence of the polypeptide of the present invention, or
a murine CXCR4 produced by bacterial cells, yeasts, animal cells
and insect cells which have been transformed with a vector
expressing a murine CXCR4 in the form of cells themselves or a
protein obtained by purifying with a usual protein chemistry
technique. The above immunogen is used to immunize an animal such
as a mouse, a rat, a hamster, a rabbit, or the like, and cells are
collected from a spleen or a lymph node to be fused with myeloma
cells, to prepare a hybridoma in accordance with a method described
in Koehler and Milstein [Nature, 256, 495497 (1975)] or a
modification thereof described in Ueda et al. [Proc. Natl. Acad
Sci. USA, 79, 4386-4390 (1982)]. The hybridoma can produce a
monoclonal antibody.
[0105] More concretely, for example, a monoclonal antibody to a
murine CXCR4 can be obtained by the following steps. [0106] (a)
immunizing a mouse with a murine CXCR4 protein; [0107] (b)
enucleating an immunized murine spleen and separating spleen cells;
[0108] (c) fusing separated spleen cells with murine myeloma cells
in the presence of a fusion enhancing agent (for example,
polyethylene glycol) in accordance with a method described in
Koehler et al. above; [0109] (d) culturing hybridoma cells obtained
in a selection medium in which non-fusion myeloma cells do not
grow; [0110] (e) selecting desired antibody-producing hybridoma
cells by means of ELISA method, an immunoelectrotransfer method,
and the like, and cloning the cells by a limiting dilution method;
and [0111] (f) collecting an anti-mouse murine CXCR4 monoclonal
antibody.
[0112] In addition, the monoclonal antibody against a fusion
protein of a murine CXCR4 with a human CXCR4/fusin/HUMSTSR is also
encompassed in the present invention.
[0113] The monoclonal antibody mentioned above can be obtained by
producing a fusion protein of a murine CXCR4 with a human
CXCR4/fusin/HUMSTSR, and performing the above techniques with the
resulting protein or a peptide thereof as an immunogen.
Pharmaceutical Composition and Cells of the Present Invention
[0114] The pharmaceutical composition of the present invention for
the use as an AIDS onset inhibitor or an HIV-1 infection inhibitor
comprises a murine PBSF/SDF-1.
[0115] The pharmaceutical composition of the present invention can
be administered orally or parenterally. In other words, the
pharmaceutical composition can be orally administered in a form
which is usually used for administration, including, for instance,
tablets, capsules, granules, powder, and the like, or alternatively
the pharmaceutical composition can be injected intramuscularly or
subcutaneously in the form of liquid, emulsion, suspension,
liposome, and the like. In addition, the pharmaceutical composition
can be administered to rectum as a suppository. These preparations
can be produced by formulating the effective ingredients of the
present invention with usual carriers, excipients, binding agents,
stabilizers, buffers, dissolution auxiliaries, isotonic agents, and
the like, which are pharmaceutically acceptable.
[0116] The dosage and the number of administration may differ
depending on symptoms, case history, ages, body weights, forms of
administration, and the like of the patients. For example, when
orally administered to an adult, it can be administered at once or
divided in several portions by appropriately adjusting the dosage
to a range of usually from 5 to 500 mg, preferably from 10 to 100
mg, per one day.
[0117] In addition, the cells of the present invention are cells
expressing the polypeptide of the present invention described
above, or cells expressing both of the above polypeptide and a
human CD4 protein.
[0118] The above cells can be obtained, for instance, by the
following method. Specifically, a vector into which a
polynucleotide encoding a murine CXCR4 is incorporated is obtained.
As the vector, any of those known per se, such as pEFBOS, pCAGGS,
and pMX can be used. Thereafter, the vector described above into
which the polynucleotide is incorporated, is introduced into cells
to be expressed, whereby obtaining the cells of the present
invention. As the cells to be expressed, there can be included a
cell line derived from a Chinese hamster ovary, CHO cells, a human
colon cancer cell line, SW480 cells, a human osteoblastsarcoma cell
line, HOS cells, a human glioblastoma cell line, U87MG cells, and
the like. In addition, a method of introducing a vector includes,
for instance, a calcium phosphate method and methods using
Lipofectin (GibcoBRL) and Lipofectamine (GibcoBRL).
[0119] Since a murine CXCR4 is found to be an HIV-1 coreceptor, the
cells of the present invention can be used to screen AIDS onset
inhibitors, HIV-1 infection inhibitors, and PBSF/SDF-I agonists and
antagonists, to detect an AIDS onset or an HIV-1 infection, and the
like.
Screening Method of the Present Invention
[0120] The screening method of the present invention includes a
method of screening AIDS onset inhibitors, HIV-1 infection
inhibitors, and murine or human PBSF/SDF-1 agonists and
antagonists. Concretely, the following methods are illustrated.
[0121] 1) A method of screening an AIDS onset inhibitor or an HIV-1
infection inhibitor, characterized in that the method comprises the
steps of: [0122] (a) mixing the cells of the present invention
described above; a human T-cell-line-tropic HIV-1; and a substance
to be screened, and incubating the resulting mixture; and [0123]
(b) analyzing localization of an HIV-1 in the cells. [0124] 2) A
method of screening an AIDS onset inhibitor or an HIV-1 infection
inhibitor, characterized in that the method comprises the steps of:
[0125] (a) mixing the cells of the present invention described
above; cells expressing an HIV-1 envelope protein; and a substance
to be screened, and incubating the resulting mixture; and [0126]
(b) determining a level of the fusion of the above cells with the
cells expressing an HIV-1 envelope protein. [0127] 3) A method of
screening an AIDS onset inhibitor or an HIV-1 infection inhibitor,
or a PBSF/SDF-1 agonist or antagonist, characterized in that the
method comprises the steps of: [0128] (a) mixing the cells of the
present invention described above; a murine or human PBSF/SDF-1;
and a substance to be screened, and incubating the resulting
mixture; and (b)determining an intracellular calcium ion level
and/or determining a binding activity of an expressed polypeptide
with the murine or human PBSF/SDF-1.
[0129] In addition, as a human T-cell-line-tropic HIV-1, there can
be included an HIV-1 IIIB strain (provided by Prof. Harada of
Kumamoto Univ.) and an HIV-1 NL432 strain (provided by Prof. Adachi
of Tokushima Univ.).
Embodiment 1
[0130] It is more preferable that the step for analyzing the
localization of an HIV-1 is carried out with a monoclonal antibody
against a human T-cell-line-tropic HIV-1.
[0131] The method of analysis using the above monoclonal antibody
is not particularly limited, and includes any known usual
method.
[0132] In addition, as the method for analyzing the localization of
an HIV-1, the following enzyme method may also be employed.
[0133] Specifically, as "the cells of the present invention" used
in this method, those preferably used are cells expressing a human
CD4 protein and a coreceptor (for instance, SW480, U87MG, HOS, and
the like) in which a gene for an enzyme (such as
.beta.-galactosidase, luciferase, or CAT) is introduced downstream
of LTR, which is an expression promoter of an HIV-1 gene. When the
cells are infected with an HIV-1, a tat protein, which is one kind
of viral proteins, is expressed which in turn activates LTR.
Accordingly, the infection level can be quantified by determining
the enzymatic activity in the cell lysate.
Embodiment 2
[0134] As the cells expressing an HIV-1 envelope protein, there can
be included, for instance, ones in which an HIV-1 envelope protein
gene is introduced into an HeLaS3. Further, ones in which, for
instance, a .beta.-galactosidase subunit (either one of .alpha. or
.omega.) gene is additionally introduced are preferably used. In
addition, as "the cells of the present invention," there can be
preferably used, for example, ones in which a human CD4 protein and
a coreceptor are introduced into an NIH3T3; and ones in which a
.beta.-galactosidase subunit (either one of .alpha. or .omega.),
and different from that introduced into the cells expressing an
HIV-1 envelope protein) gene is additionally introduced. When the
cells expressing an HIV-1 envelope protein and the cells of the
present invention are subjected to cell fusion,
.beta.-galactosidase .alpha.-subunit and .omega.-subunit are
associated to form an active .beta.-galactosidase. Accordingly, the
cell fusion level can be measured by mixing the cells of both
parties, culturing them, and then determining the galactosidase
activity in the cell lysate.
Embodiment 3
[0135] When an activity for increasing an intracellular calcium ion
level is observed as a result of the incubation in Step (a), it is
possible that the substance to be screened is an agonist. When the
binding between the substance to be screened and a receptor is
observed even though no increase in the activity for intracellular
calcium ion level is observed, it is possible that the substance to
be screened is an antagonist. In addition, when there are
influences in the activity for increasing an intracellular calcium
ion level of a murine PBSF/SDF-1 and/or on a binding activity with
a receptor, i.e., the activities are inhibited, it is possible that
the substance to be screened is an antagonist. In addition, the
above antagonist is exemplified by a hematopoetic stem cell
liberator.
Detection Kit and Detection Method
[0136] The kit for detecting an AIDS onset or an HIV-1 infection of
the present invention is characterized in that the kit comprises
the cells of the present invention.
[0137] By using the above kit, an AIDS onset or an HIV-1 infection
can readily be detected. The kit of the present invention is
intended to perform the detection using the method for detection of
the present invention described below.
[0138] The method for detecting an AIDS onset or an HIV-1 infection
of the present invention is characterized in that the method
comprises; [0139] (a) mixing the cells of the present invention
described above with sera, blood cells or blood of a patient
suspected to be infected with an HIV-1, and incubating the
resulting mixture, and [0140] (b) analyzing localization of an
HIV-I in the cells or determining a level of the fusion of the
cells with HIV-1-infected cells.
[0141] As the method for analyzing localization of an HIV-1 used
herein, there are included ones employed in an AIDS onset inhibitor
or an HIV-1 infection inhibitor of the present invention. In
addition, as the method of determining a level of the fusion of the
cells with HIV-1-infected cells, there are included ones employed
in an AIDS onset inhibitor or an HIV-1 infection inhibitor of the
present invention. Incidentally, the term "HIV-1-infected cells" in
Step (b) refers to HIV-1-infected blood cells of a patient
suspected to be infected with an HIV-1.
Utility of the Present Invention
[0142] Both of a murine CXCR4 and a human CXCR4/fusin/HUMSTSR of
the present invention react with a murine PBSF/SDF-1. Since there
is a difference of only one amino acid out of 71 amino acids in the
murine and human PBSF/SDF-1s, a murine CXCR4 is expected to be
bound also with a human PBSF/SDF-1. Since a human PBSF/SDF-I
inhibits an infection with a T-cell-line-tropic HIV-1 mediated by a
CXCR4/fusin/HUMSTSR, an antibody against the murine CXCR4 protein
of the present invention and an antibody against a chimera protein
having a binding site with a T-cell-line-tropic HIV-1 resulting
from mutual substitution of the extracellular domains of a murine
CXCR4 and a human CXCR4/fusin/HUMSTSR can be used as an HIV-1
infection inhibitor, i.e., a therapeutic agent against AIDS.
[0143] By means of the method of screening agonists and antagonists
of a chimera protein resulting from mutual substitution of the
extracellular domains of a murine CXCR4 protein, and murine CXCR4
and human CXCR4/fusin/HUMSTSR provided by the present invention,
such agonists and antagonists can be obtained, each of which can be
used as an HIV-1 infection inhibitor, i.e., a therapeutic agent
against AIDS.
[0144] The present invention will be described in further detail by
means of the following working examples, but the present invention
is by no means limited to these examples.
EXAMPLES
Example 1
Cloning of cDNA of Murine CXCR4
[0145] (1) Synthesis of Primer
[0146] Based on a known amino acid sequence of a chemokine
receptor, a condensed forward primer C2F2-2 (SEQ ID NO: 9) to a DNA
sequence encoding an amino acid sequence of a second
transmembrane-spanning domain, and a condensed reverse primer C4RI
(SEQ ID NO: 10) to a DNA sequence encoding an amino acid sequence
of a seventh transmembrane-spanning domain were synthesized using a
DNA synthesizer ("Cyclone Plus," Nippon Millipore). [0147] (2)
Purification of mRNA from Murine Pre-B-Cell Line DW34
[0148] A murine pre-B-cell line DW34 was suspended in RPMI 1640
medium. After culturing for one week, the culture was washed with
Dulbecco PBS(-) [Nissui], and mRNA was purified by using mRNA
Purification Kit (Pharmacia). [0149] (3) Cloning of cDNA Fragment
of Murine CXCR4
[0150] A single-stranded cDNA was synthesized from 200 ng of mRNA
purified from a murine pre-B-cell line DW34 with Ready-To-Go
T-Primed First-Strand Kit (Pharmacia). PCR reaction (30 cycles
under conditions of 94.degree. C. for 0.5 minutes, 55.degree. C.
for 0.5 minutes, and 72.degree.C. for 1 minute) was carried out
using the resulting single-stranded cDNA as a template, C2F2-2 and
C4RI as primers, and Taq as a thermostable DNA polymerase. The
resulting reaction mixture was separated by low-melting point
agarose gel electrophoresis, and a DNA band of a desired size
(about 690 bp) was excised, and a DNA fragment was purified by
using Wizard PCR Preps DNA Purification System (Promega). The
resulting DNA fragment was inserted into pT7Blue vector by using a
DNA Ligation Kit (Takara). The nucleotide sequence of the inserted
DNA was determined by PRISM Ready Reaction Sequence Kit (Applied
Biosystems) and DNA Sequencer (Applied Biosystems). The resulting
cDNA sequence of a murine CXCR4 is shown by SEQ ID NO: 3. Based on
the cDNA sequence of a murine CXCR4 obtained in the manner
described above, primers as shown by SEQ ID NO: 11 and as shown by
SEQ ID NO: 12 were synthesized, and a cDNA clone containing
5'-terminal was obtained using the cDNA of DW34 cells obtained as
described above as a template, with Marathon cDNA Amplification Kit
(Clontech). The resulting cDNA sequence of a murine CXCR4 is shown
by SEQ ID NO: 5.
Example 2
Expression of Murine CXCR4 in Each Tissue
[0151] (1) Preparation of Probe
[0152] In order to study the expression of a murine CXCR4 in each
murine tissue, firstly, a probe was prepared as follows. Based on
the nucleotide sequence of a murine CXCR4 gene, a DNA sequence (SEQ
ID NO: 19) in the same direction corresponding to the second
transmembrane-spanning-domain portion, and a DNA sequence (SEQ ID
NO: 20) in the opposite direction corresponding to the seventh
transmembrane-spanning-domain portion were synthesized as primers
to be used in the subsequent PCR. PCR reaction was carried out for
30 cycles under conditions of 94.degree. C. for 1 minute,
55.degree. C. for 1 minute, and 72.degree. C. for 2 minutes, using
the cDNA of the nucleotide sequence obtained in Item (3) of Example
1 above as a template, and a Taq Polymerase. The resulting reaction
mixture of the PCR was separated by agarose gel electrophoresis,
and a DNA band of a desired size (about 690 bp) was excised, and a
DNA fragment was purified by using Wizard PCR Preps DNA
Purification System. Fifty nanograms of the resulting DNA fragment
was .sup.32P-labeled by using Prime-It II Random Primer Labelling
Kit (Stratagene) to be used as a probe. [0153] (2) Northern
Blotting Analysis of Murine Tissue and Murine Fetus
[0154] mRNAs Of various murine tissues and mRNAs of murine fetuses
of 7 days, 11 days, 15 days and 17 days after embryogenesis were
separated by electrophoresis, and the transferred membrane and the
probe obtained in Item (1) above were subjected to hybridization.
The membrane was washed by immersing in 2.times.SSC containing
0.05% SDS at room temperature for 15 minutes twice, and further
immersing in 0.1.times.SSC containing 0.1% SDS at 50.degree. C. for
20minutes twice. The radioactive rays of this membrane were
detected by autoradiography. The results are shown in FIG. 2 A
(murine tissue) and B (murine fetus). It is clear from the
intensities of the bands that strong signals were obtained at
thymus, lymph node, and spleen, and week signals were obtained at
brain, small intestine, stomach, and kidney. In addition, strong
signals were obtained in the entire murine fetus.
Example 3
Cloning of Genomic DNA of Murine CXCR4
[0155] (A) Preparation of Probe
[0156] Based on the nucleotide sequence of a murine CXCR4 cDNA
obtained in Item (3) of Example 1 above, appropriate forward and
reverse primers were synthesized to be used in the subsequent PCR.
A double-stranded DNA was obtained from the cDNA in Item (3) of
Example 1, and PCR reaction was carried out using this
double-stranded DNA as a template and a Taq polymerase. The
reaction product was separated by agarose gel electrophoresis, and
a DNA band of a desired size (about 690 bp) was excised, and a DNA
fragment was purified. Fifty nanograms of the resulting DNA
fragment was .sup.32P-labeled using Prime-It II Random Primer
Labelling Kit (Stratagene) as a probe. [0157] (B) Cloning of Murine
Genomic Library
[0158] First, with a 129/SvJ murine liver genomic library
incorporated into a phage vector .lamda.FIXII was infected
Escherichia coli, and as a primary screening, and spread over a
plate to form a plaque, and the plaque was transferred to nylon
membrane (Du Pont). This membrane was pre-hybridized by immersing
the membrane in a pre-hybridization reagent [5.times.SSPE (0.9M
NaCl, 0.05M sodium phosphate at pH 7.7, 0.005M Na.sub.2EDTA), 50%
formamide, 5.times.Denhardt's reagent, 50 .mu.g/ml salmon sperm
DNA, 0.1% SDS]. Thereafter, the pre-hybridized membrane was
hybridized together with the probe obtained in Item (A) above at
42.degree. C. for 15 hours by immersing the membrane in a
hybridization reagent [5.times.SSPE, 50% formamide,
1.times.Denhardt's reagent, 10%-dextran disodium sulfate, 50
.mu.g/ml salmon sperm DNA, 0.1% SDS]. After washing the membrane,
the radioactivity was detected, and positive plaques giving signals
were selected. The selected plaques were successively diluted to
carry out secondary and tertiary screening, and whereby two single
clones were selected.
[0159] The cloned phage DNA was cleaved with various restriction
enzymes, and separated by agarose gel electrophoresis. Those bands
having the same patterns were considered to be the same clone, and
the cloned phage DNA was cleaved such that a positive band of the
size as small as possible could be obtained by repeating the same
hybridization. The selected positive DNA fragment was inserted into
a pBluescripts KSII vector, and the nucleotide sequence was
determined by dideoxy method. The resulting DNA sequence of the
murine CXCR4 gene is shown by SEQ ID NO: 7. A nucleotide sequence
containing the longest open reading frame was found from the
nucleotide sequence as shown by SEQ ID NO: 5 and the nucleotide
sequence as shown by SEQ ID NO: 7, and its nucleotide sequence is
shown by SEQ ID NO: 1. Also, this nucleotide sequence as shown by
SEQ ID NO: 1 was subjected to nucleic acid homology search with the
GenBank/EMBL/DDBJ DNA sequence data base. As a result of the
search, it was clarified that the resulting clone is a DNA encoding
a novel murine chemokine receptor, and the clone was named murine
CXCR4.
Example 4
Homology Analysis of Amino Acid Sequence of Murine CXCR4
[0160] The amino acid sequence (SEQ ID NO: 2) which was deduced
based on the nucleotide sequence of a murine CXCR4 was estimated to
be a trimer G protein-coupled receptor containing a seven
transmembrane-spanning domain, characteristic to a chemokine
receptor. The amino acid sequence thereof was compared with a known
sequence of a CXC chemokine receptor (GenBank, EMBL, DDBJ were used
as data base, and analyzed with BLAST). As a result, the analyzed
sequence most closely resembled human CXCR4/fusin/HUMSTSR (90%
identity), and homologies with monkey CXCR4 and bovine CXCR4 were
89% and 86%, respectively, and homologies with rat IL-8RB, rabbit
IL-8RA, and rabbit IL-8RB were 49%, 47%, and 45%, respectively.
Example 5
Expression of Murine CXCR4 and Human CXCR4/fusin/HUMSTSR
[0161] (1) Preparation for Expression Vectors of Murine CXCR4,
Human CCCRR2B and
[0162] Human CXCR4/fusin/HUMSTSR
[0163] In order to clone previously reported human chemokine
receptors a CC CKR2B gene and a CXCR4/fusin/HUMSTSR gene, PCR
reaction was carried out in the following manner using a cDNA of a
human monocyte cell line THP-1. Five-hundred nanograms of a cDNA of
the THP-1 cells was used as a template, the primers as shown by SEQ
ID NO: 15 and SEQ ID NO: 16 were used for amplification of human
CXCR4/fusin/HUMSTSR, and the primers as shown by SEQ ID NO: 13 and
SEQ ID NO: 14 were used for amplification of CC CKR2B, each primer
of which was used in an amount of 500 ng. As the enzyme for the
reaction, Taq Polymerase (Takara Shuzo) was used. The reaction was
carried out for I cycle at 94.degree. C. for 3 minutes; thereafter,
35 cycles at 94.degree. C. for 1 minute, 55.degree. C. for 2
minutes, and 72.degree. C. for 3 minutes; and further at 72.degree.
C. for 3 minutes. The gene fragments of human CXCR4/fusin/HUMSTSR
and CC CKR2B obtained by the reaction were each incorporated into
TA cloning sites of pCRII (Invitrogen). The plasmids obtained in
the manner described above were named pCRIICXCR4 and pCRIICC CKR2B,
respectively. Subsequently, the resulting pCRIICXCR4 and pCRIICC
CKR2B plasmids were respectively digested with NotI and XboI (both
from Takara Shuzo), and the digested fragment was incorporated into
a NotI/XboI site of pCAGGStkNeo. The plasmids obtained in the
manner described above were named pCANCXCR4 and pCANCC CKR2B,
respectively.
[0164] In order to clone the murine CXCR4 gene, PCR technique was
carried out using a single-stranded cDNA of the murine pre-B-cell
line DW34 as shown by SEQ ID NO: 5 obtained in Item (3) of Example
I above as a template. One hundred nanograms of the cDNA was used
as a template, and the primers as shown by SEQ ID NO: 17 and as
shown by SEQ ID NO: 18 were used. As the enzyme used for reaction,
ExTaq (Takara Shuzo) was used. The reaction was carried out for 1
cycle at 94.degree. C. for 3 minutes; thereafter, 20 cycles at
94.degree. C. for 1 minute, 55.degree. C. for 1 minute, and
72.degree. C. for 2 minutes; and further at 72.degree. C. for 5
minutes. The resulting murine CXCR4 gene fragment was digested with
NotI and XhoI (Takara Shuzo), and the digested fragment was
incorporated into a NotI/XboI site of pCAGGStkNeo. The plasmid
obtained in the manner described above was named pCANmPBSFR. [0165]
(2) Expression of Murine CXCR4, Human CXCR4/fusin/HUMSTSR and
Human
[0166] CC CKR2B in CHO Cells
[0167] CHO cells were cultured in a cell culture petri dish (Iwaki
Glass Co., Ltd.) having a diameter of 10 cm at 37.degree. C. for 2
day in the presence of 10% CO.sub.2 gas. Each 30 .mu.g of DNAs of
expression vectors of the three chemokine receptors obtained in
Item (1) above (pCANmPBSFR, pCANCXCR4 and pCANCC CKR2B) was
dissolved in 25 .mu.l of distilled water, and to the resulting
mixed solution was added 500 .mu.l of 250 mM calcium chloride
(nacalaitesque). To the liquid mixture of the DNA and calcium
chloride was added 500 .mu.l of 2.times.BBS solution [50 mM BES
(SIGMA), 280 mM sodium chloride (nacalaitesque) and 1.5 mM disodium
hydrogenphosphate (nacalaitesque)], and then allowed to stand at
room temperature for 25 minutes. The DNA solution prepared in the
manner described above was added dropwise to the petri dish in
which the CHO cells were cultured, and the cells were cultured at
35.degree. C. for 20 hours in the presence of 3% CO.sub.2 gas to
introduce the DNA into the cells. The cells into which the DNA was
introduced were washed twice with 3 ml of PBS(+), and thereto was
added 10 ml of .alpha.-MEM (GIBCO) containing 10% FCS solution, and
the cells were cultured at 37.degree. C. for one day in the
presence of 5% CO.sub.2 gas.
[0168] Subsequently, the cells were suspended in a medium prepared
by adding 2 mg/ml GENETICIN (Wako Pure Chemical Industries, Ltd.)
to an a-MEM (GIBCO) medium containing 10% FCS, and divided into
cell culture petri dishes (Iwaki Glass Co., Ltd.) each having a
diameter of 10 cm at a cell density of 5.times.10.sup.3 cells/petri
dish. The culture was continued at 37.degree. C. in the presence of
10% CO.sub.2 gas, and GENETICIN-resistant cells were used for
determination of an intracellular calcium level as CHO cell lines
expressing a murine CXCR4, a human CXCR4/fusin/HUMSTSR, and CC
CKR2B. As illustrated in Example 6 given below, since the CC CKR2B
was found to have an activity of increasing the intracellular
calcium level owing to the addition of a specific ligand MCP-1, the
expression of the receptor was confirmed. In addition, the murine
CXCR4 and the human CXCR4/fusin/HUMSTSR were also considered to be
similarly expressed, because transformation and culture were
carried out in the same manner using the same cell line as the CC
CKR2B.
Example 6
Biological Activity of Murine CXCR4
[0169] Each of the CHO cells expressing the murine CXCR4 and the
human chemokine receptors (CXCR4/fusin/HUMSTSR and CC CKR2B)
obtained in Item (2) of Example 5 above was washed with Dulbecco
PBS(-), and then suspended in an HBSS buffer (containing 125 mM
NaCl, 5 mM KCl, 1 mM MgCl2, 0.5 mM glucose and 0.1% BSA in 20 mM
Hepes at pH 7.4) at a cell density of 5.times.10.sup.6 cells/ml. To
the resulting suspension was further added fura-PE3AM (Texas
Fluorescence Laboratory) so as to have a concentration of 2.5
.mu.M, and the cells were then incubated at 37.degree. C. for 30
minutes. After washing with the HBSS buffer, each of the cells
expressing CC chemokine receptor was suspended in the same buffer
at a cell density of 5.times.10.sup.6 cells/ml. The changes in the
fluorescence when each chemokine (murine PBSF/SDF-I or human MCP-1)
was added to 500 .mu.l of each of the resulting suspensions of the
cells expressing CC chemokine receptor so as to have a
concentration of 100 nM were determined with a
spectrofluorophotometer (LS5OB, PERKIN-ELMER) at the excitation
wavelengths of 340 nm and 380 nm, the fluorescence wavelength of
510 nm and the response of 0.5 seconds. The results are shown in
FIG. 3 to 6 which are represented in terms of the ratio of the
fluorescence intensity at 340 nm and that at 380 nm over a passage
of a time period.
[0170] In the murine PBSF/SDF-1 stimulation, there was found to be
an increase in the ratios of fluorescence intensity in the cells
expressing a murine CXCR4 and a human CXCR4/fusin/HUMSTSR, while
there was found to be no increase in the ratios of fluorescence
intensities in the cells expressing CC CKR2B, which is a CC
chemokine receptor. Incidentally, in the stimulation with a MCP-1
peptide, which is a positive control to a receptor, there was found
to be an increase in the ratio of fluorescence intensities in the
cells expressing CC CKR2B. Therefore, the murine PBSF/SDF-1 was
found to have an activity of increasing the intracellular calcium
ion level specifically to the CHO cells expressing the murine CXCR4
and the human CXCR4/fusin/HUMSTSR according to the present
invention. Also, as shown in FIG. 7, in the cells expressing a
murine CXCR4, the desensitization was found wherein there were no
changes in the ratio of the fluorescence intensities by continuous
addition of a murine PBSF/SDF-1. The desensitization was not found
when a human MCP-1, a negative control, was added. From these
results, it was clarified that the receptor of the present
invention is the receptor of a murine PBSF/SDF-1 receptor.
Examnple 7
Materials and Methods
[0171] Cell lines: Murine NIH3T3 cells, SW480 cells derived from
human small intestine epithelium, and U87MG derived from human
gliacyte were cultured in DMEM containing 10% FCS. Human HeLaS3
cells were cultured in RPMI1640 containing 10% FCS. HOS cells
derived from human osteoblasts were cultured in Eagle MEM
containing 1% non-essential amino acids (Gibco) and 10% FCS.
[0172] Viruses: An HIV-1 NL432 strain was provided by Prof. Adachi
(Tokushima Univ.). A IIIB strain was provided by Prof. Harada
(Kumamoto Univ.). An SF162 strain was provided by Prof. J. A. Levy
(San Francisco Univ., California). HIV-1 chimera virus clones,
NL432env-162 and NL432V3-162 were provided by Isaka (Shionogi &
Co., Ltd.). Recombinant vaccinia viruses, Vac. Env (NL432 env),
Vac. Env 162 (SF162 env) and Vac T4 (CD4) were provided by Prof.
Shioda (Tokyo Univ.). LO-T7 (T7 polymerase) was provided by M.
Kohara (TORITSU RINSHOKEN).
[0173] Transfection to cells: NIH3T3 cells were cultured overnight
in a 24-well plate at a cell density of 5.times.10.sup.4 cells per
well, and transfected with a chemokine receptor gene which was
incorporated into pBluescript using Lipofectamine (Gibco). 4 Hours
after initiation of the transfection, the cells were washed with
PBS, and thereto a culture medium was added, and then cultured at
37.degree. C. overnight to be subjected to a fusion assay. The
SW480 cells and the HOS cells were cultured overnight in a 6
cm-plate at a cell density of 5.times.10.sup.5 cells. The SW480
cells were transfected with a plasmid mixture of 5 .mu.g of the
receptor gene which was incorporated into pEF-BOS, 7.5 .mu.g of
T4-Neo, a vector expressing CD4, and 2.5 .mu.g of LTR
(EcoRV)-.beta.-Gal-Neo by means of a modified calcium phosphate
method. The HOS cells constitutively expressing a human CD4 and an
LTR-Gal were transfected with 15 .mu.g of a receptor gene which was
incorporated into pEF-BOS by the same method. Each of the cells was
cultured at 35.degree. C. overnight in the presence of 3% CO.sub.2,
washed with PBS(-), and collected with PBS containing 0.5 mM EDTA.
Thereafter, the culture was plated in over a 12-well plate, and
cultured at 37.degree. C. overnight to be subjected to an infection
assay.
[0174] Cell fusion assay: In order to quantify the cell fusion, we
employed a modified cell fusion assay utilizing
.alpha.-complementation of .beta.-galactosidase (.beta.-gal) (Shida
et al., manuscript in preparation).
[0175] A .beta.-gal a-subunit and an env protein were introduced
into HeLaS3 cells (24-well plate, 1.times.10.sup.5 cells/well),
which are effector cells, by using a recombinant vaccinia
virus.
[0176] A human CD4, a .beta.-gal .omega.-subunit, and a T7 RNA
polymerase were introduced into NIH3T3 cells (24-well plate,
5.times.10.sup.4 cells/well), which are target cells, by using a
recombinant vaccinia virus, and a chemokine receptor was
transfected to the target cells using Lipofectamine. 16 Hours after
transfection, the effector cells and the target cells were washed
with PBS containing 0.5 mM CaCl.sub.2, and treated with 2D5, an
anti-vaccinia virus antibody, in order to inhibit the non-specific
cell fusion caused by the vaccinia virus. The effector cells were
suspended in a Hanks buffer (pH 7.6) containing 3 mM CaCl.sub.2,
and overlayered on the target cells in the 24-well plate.
Thereafter, the resulting overlayered mixture was centrifuged at
1,300 rpm for 5 minutes to initiate the cell fusion.
[0177] After centrifugation, the cells were cultured at 37.degree.
C. for 12 hours in the presence of 5% CO.sub.2. When the cell
fusion takes place, .alpha.-subunit and .omega.-subunit of the
.beta.-gal contained in the cytoplasmas of the fusion cells are
associated with each other to form an active .beta.-gal enzyme by
a-complementation. Therefore, after the culture medium was removed,
a solution containing 8 mM chlorophenolred-b-D-galactopyranoside
(Boehringer Mannheim), which is a .beta.-gal substrate, 45 mM
2-mercaptoethanol, 1 mM MgCl.sup.2, 100 mM Hepes at pH 8.0, 0.5%
NP40 and 0.1 mg/ml DNAse I was added in an amount of 200 .mu.l per
one well, and reacted at 37.degree. C. for 30 minutes. Thereafter,
2% SDS was added in an amount of 200 .mu.l per one well to
terminate the reaction. In order to quantify the .beta.-gal
activity in the reaction mixture, the absorbance was measured at a
wavelength of 590 nm.
[0178] Infection assay: The human SW480 or HOS cells each
expressing a human CD4 and a receptor were cultured in a 12-well
plate. To each well was added a culture containing an HIV-1 virus
(reverse transcriptase (RT) activity: SF162: 2.times.10.sup.6
RT/mL; NL432envl62, NL432V3-162 and IIIB: 5.times.10.sup.6 RT/mL;
NL432: 3.times.10.sup.6 RT/mL), and cultured at 37.degree. C. for 2
hours in the presence of 5% CO.sub.2, and then the culture medium
was added in an amount of 2.5 mL per one well. 4 Days after
infection, a Reporter lysis buffer (Promega) was added in an amount
of 400 .mu.l per one well, frozen at -80.degree. C., and then
thawed. The thawed sample was transferred to an Eppendorf tube, and
centrifuged at 12,000 rpm at 4.degree. C. for 5 minutes.
Thereafter, the .beta.-gal activity contained in the supernatant
was measured with a luminescence .beta.-gal detection kit
(Clontech).
Results
[0179] First, in order to study whether or not a murine CXCR4
supports an env-mediated cell fusion of an HIV-1, we conducted an
experiment using an assay system in which .beta.-Gal is activated
by the fusion of the effector cells (HeLaS3 cells) expressing an
env protein with the target cells (NIH3T3 cells) expressing a human
CD4 and a receptor. In this assay, the HeLaS3 cells, effector
cells, were subjected to infection with a recombinant vaccinia
virus to express an .alpha.-subunit of the .beta.-Gal and an HIV-1
env protein, and the NIH3T3 cells, target cells, were subjected to
infection with a recombinant vaccinia virus to express an
.omega.-subunit of the .beta.-Gal, a T7 polymerase and a human CD4.
After infection with a virus, the NIH3T3 cells were further
transfected with a plasmid carrying a human CXCR4, a human CCR5, or
a murine CXCR4. After an overnight culture, the effector cells and
the target cells were mixed and cultured. When the cell fusion
takes place, the .alpha.-subunit and the .omega.-subunit of the
.beta.-Gal contained in the cytoplasmas of the fusion cells are
associated with each other, whereby activating the .beta.-Gal. As
shown in FIG. 8, the HelaS3 cells expressing an env protein derived
from NL432, a T-cell-line-tropic HIV-1, are fused with the NIH3T3
cells expressing a human CXCR4 and a human CD4, but not with the
NIH3T3 cells expressing a human CCR5 and a human CD4.
[0180] Surprisingly, the HeLaS3 cells expressing an env protein
derived from NL432 are also fused with the cells expressing a
murine CXCR4 and a human CD4 similarly. The HeLaS3 cells expressing
an env protein derived from SF162, a monocyte-tropic HIV-1, are
fused with the NIH3T3 cells expressing a human CCR5 and a human
CD4, but not with the NIH3T3 cells expressing a human CXCR4 or a
murine CXCR4 and a human CD4.
[0181] Second, we have studied whether cells which expressed a
murine CXCR4 were infected with a virus. Since murine cells which
expressed human CXCR4 and CD4, including NIH3T3 cells, had a low
HIV-1 replication efficiency, three human cell lines, namely, the
SW480 cells derived from human small intestine epithelium, the HOS
cells derived from osteoblasts, and the U87MG cells derived from
human gliacytes were used as the target cells for viral infection.
These cells were transfected with a reporter gene (lacZ) having an
LTR of an HIV-1 as a promoter. When the cells are infected with a
virus, Tat protein, which is a transcription-activating factor
derived from an HIV-1, is expressed to act on an LTR, whereby
inducing the expression of lacZ. These cells were further
transfected with a human CD4 and a chemokine receptor, and then
subjected to infection with a T-cell-line-tropic virus strain
(NL432, IIIB) or a monocyte-tropic virus strain (SF162). As shown
in FIG. 9, with NL432 and IIIb were infected similarly both of
SW480 expressing a murine CXCR4 and a human CD4, and SW480
expressing a human CXCR4 and a human CD4. These findings were
consistent with the results of the fusion assay described above. On
the contrary, when the human CCR2b or CCR5 was expressed instead of
CXCR4, the cells were not infected with these viruses.
[0182] On the other hand, with SF162, the SW480 expressing a human
CCR5 and a human CD4 was infected, but cells expressing a murine
CXCR4 and a human CD4, and cells expressing a human CXCR4 and a
human CD4 were not infected therewith. In addition, similar results
were obtained even when the HOS cells or the U87MG cells were used
instead of the SW480 cells (FIG. 10 and FIG. 11). Thus, it was
suggested that a murine CXCR4 supports the entry of a
T-cell-line-tropic HIV-1 into target cells, and that it does not
affect the DNA synthesis of a provirus, the integration to a
genomic DNA or the viral expression in human cells.
[0183] Incidentally, there was clarified that the HIV-1 entry
mediated by a human CXCR4 was inhibited by a monoclonal antibody
against the V3 loop of an env protein. Accordingly, we have studied
whether the V3 loop of the env protein (gpl120) (of a
T-cell-line-tropic virus strain) is also required for a murine
CXCR4-mediated HIV-1 entry in order to confirm the function of a
murine CXCR4 is similar to that of a human CXCR4. For this purpose,
the SW480 cells expressing a human CD4 and a chemokine receptor
were subjected to infection with NL432env-162 or NL432V3-162 which
is the chimera virus clone of NL432 and SF162. As shown in FIG. 12,
NL432env-162 is a provirus clone in which the env region of a
T-cell-line-tropic virus strain NL432 is substituted with that of a
monocyte-tropic HIV-1 strain SF162, and NL432V3-162 is a provirus
clone in which the V3 loop of the env of NL432 is substituted with
that of SF162. The SW480 cells expressing a murine CXCR4 and a
human CD4 were infected with NL432, but these cells were not
infected with NL432env-162 or NL432V3-162 (FIG. 13).
[0184] On the other hand, the SW480 cells expressing a human CCR5
and a human CD4 were infected with NL432env-162 and with
NL432V3-162 (FIG. 13). It was clarified from the results that in
the case of a murine CXCR4, a V3 loop of an NL432 env is also
required for the viral entry in the same manner as the human
CXCR4.
Discussion
[0185] From the studies described above, there has been clarified
that the murine CXCR4 supports the cell membrane fusion mediated by
a T-cell-line-tropic HIV-1 env and the infection with a
T-cell-line-tropic HIV-1. These results suggest that the murine
CXCR4 is not a species-specific barrier against the infection with
an HIV-1. In the existing studies, there has been clarified even if
a human CD4 were expressed in a cell line from murine lymphocytes
or non-lymphocytes such as NIH3T3 or T-cell clone 3DT, the HIV-1 is
adsorbed but not allowed to enter. One of the interpretations of
these results is that the CXCR4 is not expressed on the surface of
the murine cells which have expressed a human CD4. In fact, a
murine PBSF/SDF-1 stimulation does not induce the change in the
intracellular calcium level in the NIH3T3 cells. Nevertheless, the
murine CXCR4 is expressed in thymocytes in which both of CD4 and
CD8 are positive as well as in thymocytes in which either CD4 or
CD8 is positive. Therefore, it is important (in order to invalidate
the interpretation described above) to determine whether or not the
3DT cells used in the experiment express CXCR4.
[0186] In a recent study, there has been clarified that a murine
homologue (murine CCR5) of a human CCR5, which is a receptor for a
monocyte-tropic HIV-1, gives no support to an HIV-1 entry. This
result suggests that there is a difference in the
species-specificity between a receptor for a monocyte-tropic HIV-1
and a receptor for a T-cell-line-tropic HIV-1. This difference may
be due to the fact that an amino acid sequence of CXCR4 is highly
conserved in between the species, as compared to those of other
chemokine receptors including CCR5. The amino acid sequence of a
murine CXCR4 has a 90% identity with that of a human CXCR4, while a
CCR5 and a CXCR2 exhibit only 82% and 71% identity, respectively,
between the murine and human sequences. This inter-species high
conservation of CXCR4 reflects the fact that PBSF/SDF-1, which is a
CXCR4 ligand, has a unique function as compared to other chemokines
such as MIP-1.alpha., MIP-.beta.and RANTES, which are CCR5 ligands.
In contrary to the understanding that the chemokines other than
PBSF/SDF-1 are involved in the chemotaxis of leukocytes in
inflammation, PBSF/SDF-1 has a function essential for a biological
development, such as hematopoiesis and cardiogenesis.
[0187] From the existing study results and from the facts that the
production efficiency of virus particles is low as compared to that
of human cells, even though a murine cell line NIH3T3 expressing a
human CD4 and a chemokine receptor supports an HIV-1 entry, the
murine cells may lack some intracellular molecules required for the
replication of an HIV-1. However, an HIV-1-infected model mouse
will be developed by generation of a transgenic mouse in which a
human gene, which is a molecule causing a species-specific barrier,
is introduced. Our results revealed the fact that it is not
necessary to introduce a human CXCR4 gene into an HIV-1-infected
model mouse. In addition, since an in vivo expression of CXCR4 is
more apt to study the initiation and the progress of the transition
from a monocyte-tropic HIV-1 to a T-cell-line-tropic HIV-1 which
leads to an onset of AIDS, there is provided valuable information
for the development of an animal model for simulation of an entire
process of an HIV-1 infection.
Industrial Applicability
[0188] According to the present invention, there can be provided a
novel murine CXC chemokine receptor gene; a polypeptide encoded by
the gene; an expression vector carrying the gene; a transformant
harboring the expression vector; a monoclonal antibody against the
polypeptide; a method for producing the polypeptide using the
transformant; and a method of screening an agonist or antagonist of
the polypeptide and also a method of screening an HIV-1 infection
inhibitor, each of which is useful in studies of a therapeutic
agent for AIDS and the functional mechanism of HIV-1 infection.
Sequence CWU 1
1
22 1 1877 DNA Mus sp. CDS (120)..(1196) 1 ccatcctaat acgactcact
atagggctcg agcggccgcc cgggcaggtg caggtagcag 60 tgaccctctg
aggcgtttgg tgctccggta accaccacgg ctgtagagcg agtgttgcc 119 atg gaa
ccg atc agt gtg agt ata tac act tct gat aac tac tct gaa 167 Met Glu
Pro Ile Ser Val Ser Ile Tyr Thr Ser Asp Asn Tyr Ser Glu 1 5 10 15
gaa gtg ggg tct gga gac tat gac tcc aac aag gaa ccc tgc ttc cgg 215
Glu Val Gly Ser Gly Asp Tyr Asp Ser Asn Lys Glu Pro Cys Phe Arg 20
25 30 gat gaa aac gtc cat ttc aat agg atc ttc ctg ccc acc atc tac
ttc 263 Asp Glu Asn Val His Phe Asn Arg Ile Phe Leu Pro Thr Ile Tyr
Phe 35 40 45 atc atc ttc ttg act ggc ata gtc ggc aat gga ttg gtg
atc ctg gtc 311 Ile Ile Phe Leu Thr Gly Ile Val Gly Asn Gly Leu Val
Ile Leu Val 50 55 60 atg ggt tac cag aag aag cta agg agc atg acg
gac aag tac cgg ctg 359 Met Gly Tyr Gln Lys Lys Leu Arg Ser Met Thr
Asp Lys Tyr Arg Leu 65 70 75 80 cac ctg tca gtg gct gac ctc ctc ttt
gtc atc aca ctc ccc ttc tgg 407 His Leu Ser Val Ala Asp Leu Leu Phe
Val Ile Thr Leu Pro Phe Trp 85 90 95 gca gtt gat gcc atg gct gac
tgg tac ttt ggg aaa ttt ttg tgt aag 455 Ala Val Asp Ala Met Ala Asp
Trp Tyr Phe Gly Lys Phe Leu Cys Lys 100 105 110 gct gtc cat atc atc
tac act gtc aac ctc tac agc agc gtt ctc atc 503 Ala Val His Ile Ile
Tyr Thr Val Asn Leu Tyr Ser Ser Val Leu Ile 115 120 125 ctg gcc ttc
atc agc ctg gac cgg tac ctc gcc att gtc cac gcc acc 551 Leu Ala Phe
Ile Ser Leu Asp Arg Tyr Leu Ala Ile Val His Ala Thr 130 135 140 aac
agt caa agg cca agg aaa ctg ctg gct gaa aag gca gtc tat gtg 599 Asn
Ser Gln Arg Pro Arg Lys Leu Leu Ala Glu Lys Ala Val Tyr Val 145 150
155 160 ggc gtc tgg atc cca gcc ctc ctc ctg act ata cct gac ttc atc
ttt 647 Gly Val Trp Ile Pro Ala Leu Leu Leu Thr Ile Pro Asp Phe Ile
Phe 165 170 175 gcc gac gtc agc cag ggg gac atc agt cag ggg gat gac
agg tac atc 695 Ala Asp Val Ser Gln Gly Asp Ile Ser Gln Gly Asp Asp
Arg Tyr Ile 180 185 190 tgt gac cgc ctt tac ccc gat agc ctg tgg atg
gtg gtg ttt caa ttc 743 Cys Asp Arg Leu Tyr Pro Asp Ser Leu Trp Met
Val Val Phe Gln Phe 195 200 205 cag cat ata atg gtg ggt ctc atc ctg
ccc ggc atc gtc atc ctc tcc 791 Gln His Ile Met Val Gly Leu Ile Leu
Pro Gly Ile Val Ile Leu Ser 210 215 220 tgt tac tgc atc atc atc tct
aag ctg tca cac tcc aag ggc cac cag 839 Cys Tyr Cys Ile Ile Ile Ser
Lys Leu Ser His Ser Lys Gly His Gln 225 230 235 240 aag cgc aag gcc
ctc aag acg aca gtc atc ctc atc cta gct ttc ttt 887 Lys Arg Lys Ala
Leu Lys Thr Thr Val Ile Leu Ile Leu Ala Phe Phe 245 250 255 gcc tgc
tgg ctg cca tat tat gtg ggg atc agc atc gac tcc ttc atc 935 Ala Cys
Trp Leu Pro Tyr Tyr Val Gly Ile Ser Ile Asp Ser Phe Ile 260 265 270
ctt ttg gga gtc atc aag caa gga tgt gac ttc gag agc att gtg cac 983
Leu Leu Gly Val Ile Lys Gln Gly Cys Asp Phe Glu Ser Ile Val His 275
280 285 aag tgg atc tcc atc aca gag gcc ctc gcc ttc ttc cac tgt tgc
ctg 1031 Lys Trp Ile Ser Ile Thr Glu Ala Leu Ala Phe Phe His Cys
Cys Leu 290 295 300 aac ccc atc ctc tat gcc ttc ctc ggg gcc aag ttc
aaa agc tct gcc 1079 Asn Pro Ile Leu Tyr Ala Phe Leu Gly Ala Lys
Phe Lys Ser Ser Ala 305 310 315 320 cag cat gca ctc aac tcc atg agc
aga ggc tcc agc ctc aag atc ctt 1127 Gln His Ala Leu Asn Ser Met
Ser Arg Gly Ser Ser Leu Lys Ile Leu 325 330 335 tcc aaa gga aag cgg
ggt gga cac tct tcc gtc tcc acg gag tca gaa 1175 Ser Lys Gly Lys
Arg Gly Gly His Ser Ser Val Ser Thr Glu Ser Glu 340 345 350 tcc tcc
agt ttt cac tcc agc taacccttat gcaaagactt atataatata 1226 Ser Ser
Ser Phe His Ser Ser 355 tatatatata tgataaagaa cttttttatg ttacacattt
tccagatata agagactgac 1286 cagtcttgta cagttttttt ttttttttaa
ttgactgttg ggagtttatg ttcctctagt 1346 ttttgtgagg tttgacttaa
tttatataaa tattgttttt tgtttgtttc atgtgaatga 1406 gcgtctaggc
aggacctgtg gccaagttct tagtagctgt ttatctgtgt gtaggactgt 1466
agaactgtag aggaagaaac tgaacattcc agaatgtgtg gtaaattgaa taaagctagc
1526 cgtgatcctc agctgttgct gcataatctc ttcattccga ggagcacccc
acccccaccc 1586 ccacccccac cccattctta aattgtttgg ttatgctgtg
tgatggtttg tttggttttt 1646 ttttgttgtt gttgttgttt tttttttctg
taaaagatgg cacttaaaac caaagcctga 1706 aatggtggta gaaatgctgg
ggtttttttt gtttgtttgt tttttcagtt ttcaagagta 1766 gattgacttc
agtccctaca aatgtacagt cttgtattac attgttaata aaagtcaatg 1826
ataaacttaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa a 1877 2 359
PRT Mus sp. 2 Met Glu Pro Ile Ser Val Ser Ile Tyr Thr Ser Asp Asn
Tyr Ser Glu 1 5 10 15 Glu Val Gly Ser Gly Asp Tyr Asp Ser Asn Lys
Glu Pro Cys Phe Arg 20 25 30 Asp Glu Asn Val His Phe Asn Arg Ile
Phe Leu Pro Thr Ile Tyr Phe 35 40 45 Ile Ile Phe Leu Thr Gly Ile
Val Gly Asn Gly Leu Val Ile Leu Val 50 55 60 Met Gly Tyr Gln Lys
Lys Leu Arg Ser Met Thr Asp Lys Tyr Arg Leu 65 70 75 80 His Leu Ser
Val Ala Asp Leu Leu Phe Val Ile Thr Leu Pro Phe Trp 85 90 95 Ala
Val Asp Ala Met Ala Asp Trp Tyr Phe Gly Lys Phe Leu Cys Lys 100 105
110 Ala Val His Ile Ile Tyr Thr Val Asn Leu Tyr Ser Ser Val Leu Ile
115 120 125 Leu Ala Phe Ile Ser Leu Asp Arg Tyr Leu Ala Ile Val His
Ala Thr 130 135 140 Asn Ser Gln Arg Pro Arg Lys Leu Leu Ala Glu Lys
Ala Val Tyr Val 145 150 155 160 Gly Val Trp Ile Pro Ala Leu Leu Leu
Thr Ile Pro Asp Phe Ile Phe 165 170 175 Ala Asp Val Ser Gln Gly Asp
Ile Ser Gln Gly Asp Asp Arg Tyr Ile 180 185 190 Cys Asp Arg Leu Tyr
Pro Asp Ser Leu Trp Met Val Val Phe Gln Phe 195 200 205 Gln His Ile
Met Val Gly Leu Ile Leu Pro Gly Ile Val Ile Leu Ser 210 215 220 Cys
Tyr Cys Ile Ile Ile Ser Lys Leu Ser His Ser Lys Gly His Gln 225 230
235 240 Lys Arg Lys Ala Leu Lys Thr Thr Val Ile Leu Ile Leu Ala Phe
Phe 245 250 255 Ala Cys Trp Leu Pro Tyr Tyr Val Gly Ile Ser Ile Asp
Ser Phe Ile 260 265 270 Leu Leu Gly Val Ile Lys Gln Gly Cys Asp Phe
Glu Ser Ile Val His 275 280 285 Lys Trp Ile Ser Ile Thr Glu Ala Leu
Ala Phe Phe His Cys Cys Leu 290 295 300 Asn Pro Ile Leu Tyr Ala Phe
Leu Gly Ala Lys Phe Lys Ser Ser Ala 305 310 315 320 Gln His Ala Leu
Asn Ser Met Ser Arg Gly Ser Ser Leu Lys Ile Leu 325 330 335 Ser Lys
Gly Lys Arg Gly Gly His Ser Ser Val Ser Thr Glu Ser Glu 340 345 350
Ser Ser Ser Phe His Ser Ser 355 3 690 DNA Mus sp. CDS (1)..(690) 3
ctg cac ctg tca gtg gct gac ctc ctc ttt gtc atc aca ctc ccc ttc 48
Leu His Leu Ser Val Ala Asp Leu Leu Phe Val Ile Thr Leu Pro Phe 1 5
10 15 tgg gca gtt gat gcc atg gct gac tgg tac ttt ggg aaa ttt ttg
tgt 96 Trp Ala Val Asp Ala Met Ala Asp Trp Tyr Phe Gly Lys Phe Leu
Cys 20 25 30 aag gct gtc cat atc atc tac act gtc aac ctc tac agc
agc gtt ctc 144 Lys Ala Val His Ile Ile Tyr Thr Val Asn Leu Tyr Ser
Ser Val Leu 35 40 45 atc ctg gcc ttc atc agc ctg gac cgg tac ctc
gcc att gtc cac gcc 192 Ile Leu Ala Phe Ile Ser Leu Asp Arg Tyr Leu
Ala Ile Val His Ala 50 55 60 acc aac agt caa agg cca agg aaa ctg
ctg gct gaa aag gca gtc tat 240 Thr Asn Ser Gln Arg Pro Arg Lys Leu
Leu Ala Glu Lys Ala Val Tyr 65 70 75 80 gtg ggc gtc tgg atc cca gcc
ctc ctc ctg act ata cct gac ttc atc 288 Val Gly Val Trp Ile Pro Ala
Leu Leu Leu Thr Ile Pro Asp Phe Ile 85 90 95 ttt gcc gac gtc agc
cag ggg gac atc agt cag ggg gat gac agg tac 336 Phe Ala Asp Val Ser
Gln Gly Asp Ile Ser Gln Gly Asp Asp Arg Tyr 100 105 110 atc tgt gac
cgc ctt tac ccc gat agc ctg tgg atg gtg gtg ttt caa 384 Ile Cys Asp
Arg Leu Tyr Pro Asp Ser Leu Trp Met Val Val Phe Gln 115 120 125 ttc
cag cat ata atg gtg ggt ctc atc ctg ccc ggc atc gtc atc ctc 432 Phe
Gln His Ile Met Val Gly Leu Ile Leu Pro Gly Ile Val Ile Leu 130 135
140 tcc tgt tac tgc atc atc atc tct aag ctg tca cac tcc aag ggc cac
480 Ser Cys Tyr Cys Ile Ile Ile Ser Lys Leu Ser His Ser Lys Gly His
145 150 155 160 cag aag cgc aag gcc ctc aag acg aca gtc atc ctc atc
cta gct ttc 528 Gln Lys Arg Lys Ala Leu Lys Thr Thr Val Ile Leu Ile
Leu Ala Phe 165 170 175 ttt gcc tgc tgg ctg cca tat tat gtg ggg atc
agc atc gac tcc ttc 576 Phe Ala Cys Trp Leu Pro Tyr Tyr Val Gly Ile
Ser Ile Asp Ser Phe 180 185 190 atc ctt ttg gga gtc atc aag caa gga
tgt gac ttc gag agc att gtg 624 Ile Leu Leu Gly Val Ile Lys Gln Gly
Cys Asp Phe Glu Ser Ile Val 195 200 205 cac aag tgg atc tcc atc aca
gag gcc ctc gcc ttc ttc cac tgt tgc 672 His Lys Trp Ile Ser Ile Thr
Glu Ala Leu Ala Phe Phe His Cys Cys 210 215 220 ctg aac ccc atc ctc
tat 690 Leu Asn Pro Ile Leu Tyr 225 230 4 230 PRT Mus sp. 4 Leu His
Leu Ser Val Ala Asp Leu Leu Phe Val Ile Thr Leu Pro Phe 1 5 10 15
Trp Ala Val Asp Ala Met Ala Asp Trp Tyr Phe Gly Lys Phe Leu Cys 20
25 30 Lys Ala Val His Ile Ile Tyr Thr Val Asn Leu Tyr Ser Ser Val
Leu 35 40 45 Ile Leu Ala Phe Ile Ser Leu Asp Arg Tyr Leu Ala Ile
Val His Ala 50 55 60 Thr Asn Ser Gln Arg Pro Arg Lys Leu Leu Ala
Glu Lys Ala Val Tyr 65 70 75 80 Val Gly Val Trp Ile Pro Ala Leu Leu
Leu Thr Ile Pro Asp Phe Ile 85 90 95 Phe Ala Asp Val Ser Gln Gly
Asp Ile Ser Gln Gly Asp Asp Arg Tyr 100 105 110 Ile Cys Asp Arg Leu
Tyr Pro Asp Ser Leu Trp Met Val Val Phe Gln 115 120 125 Phe Gln His
Ile Met Val Gly Leu Ile Leu Pro Gly Ile Val Ile Leu 130 135 140 Ser
Cys Tyr Cys Ile Ile Ile Ser Lys Leu Ser His Ser Lys Gly His 145 150
155 160 Gln Lys Arg Lys Ala Leu Lys Thr Thr Val Ile Leu Ile Leu Ala
Phe 165 170 175 Phe Ala Cys Trp Leu Pro Tyr Tyr Val Gly Ile Ser Ile
Asp Ser Phe 180 185 190 Ile Leu Leu Gly Val Ile Lys Gln Gly Cys Asp
Phe Glu Ser Ile Val 195 200 205 His Lys Trp Ile Ser Ile Thr Glu Ala
Leu Ala Phe Phe His Cys Cys 210 215 220 Leu Asn Pro Ile Leu Tyr 225
230 5 685 DNA Mus sp. CDS (120)..(683) 5 ccatcctaat acgactcact
atagggctcg agcggccgcc cgggcaggtg caggtagcag 60 tgaccctctg
aggcgtttgg tgctccggta accaccacgg ctgtagagcg agtgttgcc 119 atg gaa
ccg atc agt gtg agt ata tac act tct gat aac tac tct gaa 167 Met Glu
Pro Ile Ser Val Ser Ile Tyr Thr Ser Asp Asn Tyr Ser Glu 1 5 10 15
gaa gtg ggg tct gga gac tat gac tcc aac aag gaa ccc tgc ttc cgg 215
Glu Val Gly Ser Gly Asp Tyr Asp Ser Asn Lys Glu Pro Cys Phe Arg 20
25 30 gat gaa aac gtc cat ttc aat agg atc ttc ctg ccc acc atc tac
ttc 263 Asp Glu Asn Val His Phe Asn Arg Ile Phe Leu Pro Thr Ile Tyr
Phe 35 40 45 atc atc ttc ttg act ggc ata gtc ggc aat gga ttg gtg
atc ctg gtc 311 Ile Ile Phe Leu Thr Gly Ile Val Gly Asn Gly Leu Val
Ile Leu Val 50 55 60 atg ggt tac cag aag aag cta agg agc atg acg
gac aag tac cgg ctg 359 Met Gly Tyr Gln Lys Lys Leu Arg Ser Met Thr
Asp Lys Tyr Arg Leu 65 70 75 80 cac ctg tca gtg gct gac ctc ctc ttt
gtc atc aca ctc ccc ttc tgg 407 His Leu Ser Val Ala Asp Leu Leu Phe
Val Ile Thr Leu Pro Phe Trp 85 90 95 gca gtt gat gcc atg gct gac
tgg tac ttt ggg aaa ttt ttg tgt aag 455 Ala Val Asp Ala Met Ala Asp
Trp Tyr Phe Gly Lys Phe Leu Cys Lys 100 105 110 gct gtc cat atc atc
tac act gtc aac ctc tac agc agc gtt ctc atc 503 Ala Val His Ile Ile
Tyr Thr Val Asn Leu Tyr Ser Ser Val Leu Ile 115 120 125 ctg gcc ttc
atc agc ctg gac cgg tac ctc gcc att gtc cac gcc acc 551 Leu Ala Phe
Ile Ser Leu Asp Arg Tyr Leu Ala Ile Val His Ala Thr 130 135 140 aac
agt caa agg cca agg aaa ctg ctg gct gaa aag gca gtc tat gtg 599 Asn
Ser Gln Arg Pro Arg Lys Leu Leu Ala Glu Lys Ala Val Tyr Val 145 150
155 160 ggc gtc tgg atc cca gcc ctc ctc ctg act ata cct gac ttc atc
ttt 647 Gly Val Trp Ile Pro Ala Leu Leu Leu Thr Ile Pro Asp Phe Ile
Phe 165 170 175 gcc gac gtc agc cag ggg gac atc agt cag ggg gat ga
685 Ala Asp Val Ser Gln Gly Asp Ile Ser Gln Gly Asp 180 185 6 188
PRT Mus sp. 6 Met Glu Pro Ile Ser Val Ser Ile Tyr Thr Ser Asp Asn
Tyr Ser Glu 1 5 10 15 Glu Val Gly Ser Gly Asp Tyr Asp Ser Asn Lys
Glu Pro Cys Phe Arg 20 25 30 Asp Glu Asn Val His Phe Asn Arg Ile
Phe Leu Pro Thr Ile Tyr Phe 35 40 45 Ile Ile Phe Leu Thr Gly Ile
Val Gly Asn Gly Leu Val Ile Leu Val 50 55 60 Met Gly Tyr Gln Lys
Lys Leu Arg Ser Met Thr Asp Lys Tyr Arg Leu 65 70 75 80 His Leu Ser
Val Ala Asp Leu Leu Phe Val Ile Thr Leu Pro Phe Trp 85 90 95 Ala
Val Asp Ala Met Ala Asp Trp Tyr Phe Gly Lys Phe Leu Cys Lys 100 105
110 Ala Val His Ile Ile Tyr Thr Val Asn Leu Tyr Ser Ser Val Leu Ile
115 120 125 Leu Ala Phe Ile Ser Leu Asp Arg Tyr Leu Ala Ile Val His
Ala Thr 130 135 140 Asn Ser Gln Arg Pro Arg Lys Leu Leu Ala Glu Lys
Ala Val Tyr Val 145 150 155 160 Gly Val Trp Ile Pro Ala Leu Leu Leu
Thr Ile Pro Asp Phe Ile Phe 165 170 175 Ala Asp Val Ser Gln Gly Asp
Ile Ser Gln Gly Asp 180 185 7 1694 DNA Mus sp. CDS (1)..(1056) 7
ata tac act tct gat aac tac tct gaa gaa gtg ggg tct gga gac tat 48
Ile Tyr Thr Ser Asp Asn Tyr Ser Glu Glu Val Gly Ser Gly Asp Tyr 1 5
10 15 gac tcc aac aag gaa ccc tgc ttc cgg gat gaa aac gtc cat ttc
aat 96 Asp Ser Asn Lys Glu Pro Cys Phe Arg Asp Glu Asn Val His Phe
Asn 20 25 30 agg atc ttc ctg ccc acc atc tac ttc atc atc ttc ttg
act ggc ata 144 Arg Ile Phe Leu Pro Thr Ile Tyr Phe Ile Ile Phe Leu
Thr Gly Ile 35 40 45 gtc ggc aat gga ttg gtg atc ctg gtc atg ggt
tac cag aag aag cta 192 Val Gly Asn Gly Leu Val Ile Leu Val Met Gly
Tyr Gln Lys Lys Leu 50 55 60 agg agc atg acg gac aag tac cgg ctg
cac ctg tca gtg gct gac ctc 240 Arg Ser Met Thr Asp Lys Tyr Arg Leu
His Leu Ser Val Ala Asp Leu 65 70 75 80 ctc ttt gtc atc aca ctc ccc
ttc tgg gca gtt gat gcc atg gct gac 288 Leu Phe Val Ile Thr Leu Pro
Phe Trp Ala Val Asp Ala Met Ala Asp 85 90 95 tgg tac ttt ggg aaa
ttt ttg tgt aag gct gtc cat atc atc tac act 336 Trp Tyr Phe Gly Lys
Phe Leu Cys Lys Ala Val His Ile Ile Tyr Thr 100 105 110 gtc aac ctc
tac agc agc gtt ctc atc ctg gcc ttc atc agc ctg gac 384 Val Asn Leu
Tyr Ser Ser Val Leu Ile Leu Ala Phe Ile Ser Leu Asp 115 120 125 cgg
tac ctc gcc att gtc cac gcc acc aac agt caa agg cca agg aaa 432 Arg
Tyr Leu Ala Ile Val His Ala Thr Asn Ser Gln Arg Pro Arg Lys 130 135
140 ctg ctg gct gaa aag gca gtc tat gtg ggc gtc tgg
atc cca gcc ctc 480 Leu Leu Ala Glu Lys Ala Val Tyr Val Gly Val Trp
Ile Pro Ala Leu 145 150 155 160 ctc ctg act ata cct gac ttc atc ttt
gcc gac gtc agc cag ggg gac 528 Leu Leu Thr Ile Pro Asp Phe Ile Phe
Ala Asp Val Ser Gln Gly Asp 165 170 175 atc agt cag ggg gat gac agg
tac atc tgt gac cgc ctt tac ccc gat 576 Ile Ser Gln Gly Asp Asp Arg
Tyr Ile Cys Asp Arg Leu Tyr Pro Asp 180 185 190 agc ctg tgg atg gtg
gtg ttt caa ttc cag cat ata atg gtg ggt ctc 624 Ser Leu Trp Met Val
Val Phe Gln Phe Gln His Ile Met Val Gly Leu 195 200 205 atc ctg ccc
ggc atc gtc atc ctc tcc tgt tac tgc atc atc atc tct 672 Ile Leu Pro
Gly Ile Val Ile Leu Ser Cys Tyr Cys Ile Ile Ile Ser 210 215 220 aag
ctg tca cac tcc aag ggc cac cag aag cgc aag gcc ctc aag acg 720 Lys
Leu Ser His Ser Lys Gly His Gln Lys Arg Lys Ala Leu Lys Thr 225 230
235 240 aca gtc atc ctc atc cta gct ttc ttt gcc tgc tgg ctg cca tat
tat 768 Thr Val Ile Leu Ile Leu Ala Phe Phe Ala Cys Trp Leu Pro Tyr
Tyr 245 250 255 gtg ggg atc agc atc gac tcc ttc atc ctt ttg gga gtc
atc aag caa 816 Val Gly Ile Ser Ile Asp Ser Phe Ile Leu Leu Gly Val
Ile Lys Gln 260 265 270 gga tgt gac ttc gag agc att gtg cac aag tgg
atc tcc atc aca gag 864 Gly Cys Asp Phe Glu Ser Ile Val His Lys Trp
Ile Ser Ile Thr Glu 275 280 285 gcc ctc gcc ttc ttc cac tgt tgc ctg
aac ccc atc ctc tat gcc ttc 912 Ala Leu Ala Phe Phe His Cys Cys Leu
Asn Pro Ile Leu Tyr Ala Phe 290 295 300 ctc ggg gcc aag ttc aaa agc
tct gcc cag cat gca ctc aac tcc atg 960 Leu Gly Ala Lys Phe Lys Ser
Ser Ala Gln His Ala Leu Asn Ser Met 305 310 315 320 agc aga ggc tcc
agc ctc aag atc ctt tcc aaa gga aag cgg ggt gga 1008 Ser Arg Gly
Ser Ser Leu Lys Ile Leu Ser Lys Gly Lys Arg Gly Gly 325 330 335 cac
tct tcc gtc tcc acg gag tca gaa tcc tcc agt ttt cac tcc agc 1056
His Ser Ser Val Ser Thr Glu Ser Glu Ser Ser Ser Phe His Ser Ser 340
345 350 taacccttat gcaaagactt atataatata tatatatata tgataaagaa
cttttttatg 1116 ttacacattt tccagatata agagactgac cagtcttgta
cagttttttt ttttttttaa 1176 ttgactgttg ggagtttatg ttcctctagt
ttttgtgagg tttgacttaa tttatataaa 1236 tattgttttt tgtttgtttc
atgtgaatga gcgtctaggc aggacctgtg gccaagttct 1296 tagtagctgt
ttatctgtgt gtaggactgt agaactgtag aggaagaaac tgaacattcc 1356
agaatgtgtg gtaaattgaa taaagctagc cgtgatcctc agctgttgct gcataatctc
1416 ttcattccga ggagcacccc acccccaccc ccacccccac cccattctta
aattgtttgg 1476 ttatgctgtg tgatggtttg tttggttttt ttttgttgtt
gttgttgttt tttttttctg 1536 taaaagatgg cacttaaaac caaagcctga
aatggtggta gaaatgctgg ggtttttttt 1596 gtttgtttgt tttttcagtt
ttcaagagta gattgacttc agtccctaca aatgtacagt 1656 cttgtattac
attgttaata aaagtcaatg ataaactt 1694 8 352 PRT Mus sp. 8 Ile Tyr Thr
Ser Asp Asn Tyr Ser Glu Glu Val Gly Ser Gly Asp Tyr 1 5 10 15 Asp
Ser Asn Lys Glu Pro Cys Phe Arg Asp Glu Asn Val His Phe Asn 20 25
30 Arg Ile Phe Leu Pro Thr Ile Tyr Phe Ile Ile Phe Leu Thr Gly Ile
35 40 45 Val Gly Asn Gly Leu Val Ile Leu Val Met Gly Tyr Gln Lys
Lys Leu 50 55 60 Arg Ser Met Thr Asp Lys Tyr Arg Leu His Leu Ser
Val Ala Asp Leu 65 70 75 80 Leu Phe Val Ile Thr Leu Pro Phe Trp Ala
Val Asp Ala Met Ala Asp 85 90 95 Trp Tyr Phe Gly Lys Phe Leu Cys
Lys Ala Val His Ile Ile Tyr Thr 100 105 110 Val Asn Leu Tyr Ser Ser
Val Leu Ile Leu Ala Phe Ile Ser Leu Asp 115 120 125 Arg Tyr Leu Ala
Ile Val His Ala Thr Asn Ser Gln Arg Pro Arg Lys 130 135 140 Leu Leu
Ala Glu Lys Ala Val Tyr Val Gly Val Trp Ile Pro Ala Leu 145 150 155
160 Leu Leu Thr Ile Pro Asp Phe Ile Phe Ala Asp Val Ser Gln Gly Asp
165 170 175 Ile Ser Gln Gly Asp Asp Arg Tyr Ile Cys Asp Arg Leu Tyr
Pro Asp 180 185 190 Ser Leu Trp Met Val Val Phe Gln Phe Gln His Ile
Met Val Gly Leu 195 200 205 Ile Leu Pro Gly Ile Val Ile Leu Ser Cys
Tyr Cys Ile Ile Ile Ser 210 215 220 Lys Leu Ser His Ser Lys Gly His
Gln Lys Arg Lys Ala Leu Lys Thr 225 230 235 240 Thr Val Ile Leu Ile
Leu Ala Phe Phe Ala Cys Trp Leu Pro Tyr Tyr 245 250 255 Val Gly Ile
Ser Ile Asp Ser Phe Ile Leu Leu Gly Val Ile Lys Gln 260 265 270 Gly
Cys Asp Phe Glu Ser Ile Val His Lys Trp Ile Ser Ile Thr Glu 275 280
285 Ala Leu Ala Phe Phe His Cys Cys Leu Asn Pro Ile Leu Tyr Ala Phe
290 295 300 Leu Gly Ala Lys Phe Lys Ser Ser Ala Gln His Ala Leu Asn
Ser Met 305 310 315 320 Ser Arg Gly Ser Ser Leu Lys Ile Leu Ser Lys
Gly Lys Arg Gly Gly 325 330 335 His Ser Ser Val Ser Thr Glu Ser Glu
Ser Ser Ser Phe His Ser Ser 340 345 350 9 20 DNA Artificial
Sequence Description of Artificial Sequence Synthetic DNA 9
ctsmgtttgk cmntnkcyga 20 10 26 DNA Artificial Sequence Description
of Artificial Sequence Synthetic DNA 10 tagaksanng grttsanrca
rcagtg 26 11 25 DNA Artificial Sequence Description of Artificial
Sequence Synthetic DNA 11 tcatccccct gactgatgtc cccct 25 12 27 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
DNA 12 ccatcctaat acgactcact atagggc 27 13 30 DNA Artificial
Sequence Description of Artificial Sequence Synthetic DNA 13
cgcgtcgacc acaacatgct gtccacatca 30 14 30 DNA Artificial Sequence
Description of Artificial Sequence Synthetic DNA 14 cgctctagat
tataaaccag ccgagacttc 30 15 29 DNA Artificial Sequence Description
of Artificial Sequence Synthetic DNA 15 cgcgtcgacg ttaccatgga
ggggatcag 29 16 32 DNA Artificial Sequence Description of
Artificial Sequence Synthetic DNA 16 cgcgcggccg cttagctgga
gtgaaaactt ga 32 17 27 DNA Artificial Sequence Description of
Artificial Sequence Synthetic DNA 17 tagcggccgc gttgccatgg aaccgat
27 18 27 DNA Artificial Sequence Description of Artificial Sequence
Synthetic DNA 18 gcgtcgacta agggttagct ggagtga 27 19 20 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
DNA 19 ctgcacctgt cagtggctga 20 20 27 DNA Artificial Sequence
Description of Artificial Sequence Synthetic DNA 20 tagatgaggg
ggattgagac aacagtg 27 21 1776 DNA Mus musculus CDS (59)..(325) 21
ctcggtgtcc tcttgctgtc cagctctgca gcctccggcg cgccctcccg cccacgcc 58
atg gac gcc aag gtc gtc gcc gtg ctg gcc ctg gtg ctg gcc gcg ctc 106
Met Asp Ala Lys Val Val Ala Val Leu Ala Leu Val Leu Ala Ala Leu 1 5
10 15 tgc atc agt gac ggt aaa cca gtc agc ctg agc tac cga tgc ccc
tgc 154 Cys Ile Ser Asp Gly Lys Pro Val Ser Leu Ser Tyr Arg Cys Pro
Cys 20 25 30 cgg ttc ttc gag agc cac atc gcc aga gcc aac gtc aag
cat ctg aaa 202 Arg Phe Phe Glu Ser His Ile Ala Arg Ala Asn Val Lys
His Leu Lys 35 40 45 atc ctc aac act cca aac tgt gcc ctt cag att
gtt gca cgg ctg aag 250 Ile Leu Asn Thr Pro Asn Cys Ala Leu Gln Ile
Val Ala Arg Leu Lys 50 55 60 aac aac aac aga caa gtg tgc att gac
ccg aaa tta aag tgg atc caa 298 Asn Asn Asn Arg Gln Val Cys Ile Asp
Pro Lys Leu Lys Trp Ile Gln 65 70 75 80 gag tac ctg gag aaa gct tta
aac aag taagcacaac agcccaaagg 345 Glu Tyr Leu Glu Lys Ala Leu Asn
Lys 85 actttccagt agacccccga ggaaggctga catccgtggg agatgcaagg
gcagtggtgg 405 ggaggagggc ctgaaccctg gccaggatgg ccggcgggac
agcactgact ggggtcatgc 465 taaggtttgc cagcataaag acactccgcc
atagcatatg gtacgatatt gcagcttata 525 ttcatccctg ccctcgcccg
tgcacaatgg agcttttata actggggttt ttctaaggaa 585 ttgtattacc
ctaaccagtt agcttcatcc ccattctcct catcctcatc ttcattttaa 645
aaagcagtga ttacttcaag ggctgtattc agtttgcttt ggagcttctc tttgccctgg
705 ggcctctggg cacagttata gacggtggct ttgcagggag ccctagagag
aaaccttcca 765 ccagagcaga gtccgaggaa cgctgcaggg cttgtcctgc
agggggcgct cctcgacaga 825 tgccttgtcc tgagtcaaca caagatccgg
cagagggagg ctcctttatc cagttcagtg 885 ccagggtcgg gaagcttcct
ttagaagtga tccctgaagc tgtgctcaga gaccctttcc 945 tagccgttcc
tgctctctgc ttgcctccaa acgcatgctt catctgactt ccgcttctca 1005
cctctgtagc ctgacggacc aatgctgcaa tggaagggag gagagtgatg tggggtgccc
1065 cctccctctc ttccctttgc tttcctctca cttgggccct ttgtgagatt
tttctttggc 1125 ctcctgtaga atggagccag accatcctgg ataatgtgag
aacatgccta gatttaccca 1185 caaaacacaa gtctgagaat taatcataaa
cggaagttta aatgaggatt tggactttgg 1245 taattgtccc tgagtcctat
atatttcaac agtggctcta tgggctctga tcgaatatca 1305 gtgatgaaaa
taataataat aataataata acgaataagc cagaatcttg ccatgaagcc 1365
acagtgggga ttctgggttc caatcagaaa tggagacaag ataaaacttg catacattct
1425 tacgatcaca gacggccctg gtggtttttg gtaactattt acaaggcatt
tttttacata 1485 tatttttgtg cactttttat gtttctttgg aagacaaatg
tatttcagaa tatatttgta 1545 gtcaattcat atatttgaag tggagccata
gtaatgccag tagatatctc tatgatcttg 1605 agctactggc aacttgtaaa
gaaatatata tgacatataa atgtattgta gctttccggt 1665 gtcagccacg
gtgtattttt ccacttggaa tgaaattgta tcaactgtga cattatatgc 1725
actagcaata aaatgctaat tgtttcatgc tgtaaaaaaa aaaaaaaaaa a 1776 22 89
PRT Mus musculus 22 Met Asp Ala Lys Val Val Ala Val Leu Ala Leu Val
Leu Ala Ala Leu 1 5 10 15 Cys Ile Ser Asp Gly Lys Pro Val Ser Leu
Ser Tyr Arg Cys Pro Cys 20 25 30 Arg Phe Phe Glu Ser His Ile Ala
Arg Ala Asn Val Lys His Leu Lys 35 40 45 Ile Leu Asn Thr Pro Asn
Cys Ala Leu Gln Ile Val Ala Arg Leu Lys 50 55 60 Asn Asn Asn Arg
Gln Val Cys Ile Asp Pro Lys Leu Lys Trp Ile Gln 65 70 75 80 Glu Tyr
Leu Glu Lys Ala Leu Asn Lys 85
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